Dipeptoid prodrugs and their use

ABSTRACT

The present application relates to prodrug derivatives of 2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxyethoxy)phenyl]pyridine-3,5-dicarbonitrile, processes for their preparation, their use for the treatment and/or prophylaxis of diseases, and their use for the manufacture of medicaments for the treatment and/or prophylaxis of diseases, especially of cardiovascular disorders.

The present application relates to prodrug derivatives of2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxyethoxy)phenyl]pyridine-3,5-dicarbonitrile,processes for their preparation, their use for the treatment and/orprophylaxis of diseases, and their use for the manufacture ofmedicaments for the treatment and/or prophylaxis of diseases, especiallyof cardiovascular disorders.

Prodrugs are derivatives of an active ingredient which undergo in vivoan enzymatic and/or chemical biotransformation in one or more stagesbefore the actual active ingredient is liberated. A prodrug residue isordinarily used in order to improve the profile of properties of theunderlying active ingredient [P. Ettmayer et al., J. Med. Chem. 47,2393-2404 (2004)]. In order to achieve an optimal profile of effects itis necessary in this connection for the design of the prodrug residue aswell as the desired mechanism of liberation to conform very accuratelywith the individual active ingredient, the indication, the site ofaction and the administration route. A large number of medicaments isadministered as prodrugs which exhibit an improved bioavailability bycomparison with the underlying active ingredient, for example achievedby improving the physicochemical profile, specifically the solubility,the active or passive absorption properties or the tissue-specificdistribution. An example which may be mentioned from the wide-rangingliterature on prodrugs is: H. Bundgaard (Ed.), Design of Prodrugs:Bioreversible derivatives for various functional groups and chemicalentities, Elsevier Science Publishers B.V., 1985.

Adenosine, a purine nucleoside, is present in all cells and is releasedunder a large number of physiological and pathophysiological stimuli.Adenosine is produced inside cells on degradation of adenosine5′-monophosphate (AMP) and S-adenosylhomocysteine as intermediate, butcan be released from the cell and then exerts, by binding to specificreceptors, effects as hormone-light substance or neurotransmitter.Essential functions in particular in excitable and/or working cells invarious tissues are influenced by adenosine A1 receptors [cf. K. A.Jacobson and Z.-G. Gao, Nat. Rev. Drug Discover. 5, 247-264 (2006)].

The compound2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxy-ethoxy)phenyl]pyridine-3,5-dicarbonitrile[compound (A)] is an orally active adenosine A1 receptor agonist and iscurrently undergoing in-depth clinical testing as a possible novelactive pharmaceutical ingredient for the prevention and therapy inparticular of cardiovascular disorders [WHO Drug Information Vol. 20,No. 2 (2006); for preparation and use, see WO 03/053441, example 6].

However, compound (A) has only a limited solubility in water,physiological media and organic solvents, and an only lowbioavailability after oral administration of a suspension of crystallinematerial. On the one hand, this allows intravenous administration of theactive ingredient only in very low dosages; infusion solutions based onphysiological saline solutions can be produced only with difficulty withconventional solubilizers. On the other hand formulation in tablet formis difficult. It was therefore an object of the present invention toidentify derivatives or prodrugs of compound (A) which have an improvedsolubility in the media mentioned and/or an improved bioavailabilityafter oral administration and, at the same time, allow controlledliberation of the active ingredient (A) in the patient's body afteradministration. In addition, further areas of therapeutic use of thisactive ingredient could be opened up by an improved possibility ofintravenous administration.

A review of prodrug derivatives based on carboxylic esters and possibleproperties of such compounds is given for example in K. Beaumont et al.,Curr. Drug Metab. 4, 461-485 (2003).

The present invention relates to compounds of the general formula (I)

in which

R^(A) is a group of the formula

in which

* means the point of linkage to the O atom,

L¹ and L² are independently of one another a bond or —CH₂—,

R¹, R² and R³ are independently of one another hydrogen or methyl,

R⁴ and R⁶ are identical or different and are independently of oneanother hydrogen or the side group of a natural α-amino acid or itshomologs or isomers,

R⁵ and R⁷ are independently of one another hydrogen or methyl,

L³ is straight-chain or branched (C₂-C₄)-alkanediyl which isadditionally substituted by amino,

R⁸, R⁹ and R¹⁰ are independently of one another hydrogen or methyl,

m is the number 2, 3, 4, 5 or 6,

L⁴ is straight-chain or branched (C₂-C₄)-alkanediyl, which isadditionally substituted by carboxyl,

R¹¹ is hydrogen or methyl, and

n is the number 1, 2, 3 or 4,

and the salts, solvates and solvates of the salts thereof.

Compounds according to the invention are the compounds of the formula(I) and the salts, solvates and solvates of the salts thereof, thecompounds which are encompassed by formula (I) and are of the formulaementioned hereinafter, and the salts, solvates and solvates of the saltsthereof, and the compounds which are encompassed by formula (I) and arementioned hereinafter as exemplary embodiments, and the salts, solvatesand solvates of the salts thereof, insofar as the compounds encompassedby formula (I) and mentioned hereinafter are not already salts, solvatesand solvates of the salts.

The compounds according to the invention may, depending on theirstructure, exist in stereoisomeric forms (enantiomers, diastereomers).The invention therefore relates to the enantiomers or diastereomers andrespective mixtures thereof. The stereoisomerically pure constituentscan be isolated in a known manner from such mixtures of enantiomersand/or diastereomers.

Where the compounds according to the invention can occur in tautomericforms, the present invention encompasses all tautomeric forms.

Salts preferred for the purposes of the present invention arephysiologically acceptable salts of the compounds according to theinvention. However, salts which are themselves unsuitable forpharmaceutical applications but can be used for example for isolating orpurifying the compounds according to the invention are also encompassed.

Besides monosalts, the present invention also includes where appropriatepossible polysalts such as di- or trisalts.

Physiologically acceptable salts of the compounds according to theinvention include acid addition salts of mineral acids, carboxylic acidsand sulphonic acids, e.g. salts of hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonicacid, toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonicacid, acetic acid, trifluoroacetic acid, propionic acid, lactic acid,tartaric acid, malic acid, citric acid, fumaric acid, maleic acid andbenzoic acid.

Physiologically acceptable salts of the compounds according to theinvention also include salts of usual bases such as, by way of exampleand preferably, alkali metal salts (e.g. sodium and potassium salts),alkaline earth metal salts (e.g. calcium and magnesium salts) andammonium salts, derived from ammonia or organic amines having 1 to 16 Catoms, such as, by way of example and preferably, ethylamine,diethylamine, triethylamine, ethyldiisopropylamine, monoethanolamine,diethanolamine, triethanolamine, choline, dicyclohexylamine,dimethylaminoethanol, procain, dibenzylamine, morpholine,N-methylmorpholine, arginine, lysine, ethylenediamine, piperidine andN-methylpiperidine.

Solvates refer for the purposes of the invention to those forms of thecompounds according to the invention which form a complex in the solidor liquid state through coordination with solvent molecules. Hydratesare a specific form of solvates in which the coordination takes placewith water. Solvates preferred in the context of the present inventionare hydrates.

In the context of the present invention, the substituents have thefollowing meaning unless otherwise specified:

(C₂-C₄)-Alkanediyl is in the context of the invention a straight-chainor branched divalent alkyl radical having 2 to 4 carbon atoms. Astraight-chain alkanediyl radical having 2 to 4 carbon atoms ispreferred. Examples which may be preferably mentioned are:ethane-1,2-diyl (1,2-ethylene), ethane-1,1-diyl, propane-1,3-diyl(1,3-propylene), propane-1,1-diyl, propane-1,2-diyl, propane-2,2-diyl,butane-1,4-diyl (1,4-butylene), butane-1,2-diyl, butane-1,3-diyl,butane-2,3-diyl. The alkanediyl radical is additionally substituted inthe case of the group L³ by an amino group and in the case of the groupL⁴ by a carboxyl group.

The side group of an α-amino acid in the meaning of R⁴ and R⁶encompasses both the side groups of naturally occurring α-amino acidsand the side groups of homologs and isomers of these α-amino acids. Theα-amino acid may in this connection have both the L and the Dconfiguration or else be a mixture of the L form and D form. Examples ofside groups which may be mentioned are: methyl (alanine), propan-2-yl(valine), propan-1-yl (norvaline), 2-methylpropan-1-yl (leucine),1-methylpropan-1-yl (isoleucine), butan-1-yl (norleucine),tert-butyl(2-tert-butylglycine), phenyl (2-phenylglycine), benzyl(phenylalanine), p-hydroxybenzyl (tyrosine), indol-3-ylmethyl(tryptophan), imidazol-4-ylmethyl (histidine), hydroxymethyl (serine),2-hydroxyethyl (homoserine), 1-hydroxyethyl (threonine), mercaptomethyl(cysteine), methylthiomethyl (S-methylcysteine), 2-mercaptoethyl(homocysteine), 2-methylthioethyl (methionine), carbamoylmethyl(asparagine), 2-carbamoylethyl (glutamine), carboxymethyl (asparticacid), 2-carboxyethyl (glutamic acid), 4-aminobutan-1-yl (lysine),4-amino-3-hydroxybutan-1-yl (hydroxylysine), 3-aminopropan-1-yl(ornithine), 3-guanidinopropan-1-yl (arginine), 3-ureidopropan-1-yl(citrulline). Preferred α-amino acid side groups in the meaning of R⁴are methyl (alanine), propan-2-yl (valine), propan-1-yl (norvaline),2-methylpropan-1-yl (leucine), 1-methylpropan-1-yl (isoleucine),butan-1-yl (norleucine), benzyl (phenylalanine), p-hydroxybenzyl(tyrosine), imidazol-4-ylmethyl (histidine), hydroxymethyl (serine),1-hydroxyethyl (threonine), carbamoylmethyl (asparagine),2-carbamoylethyl (glutamine). Preferred α-amino acid side groups in themeaning of R⁶ are imidazol-4-ylmethyl (histidine), 4-aminobutan-1-yl(lysine), 3-aminopropan-1-yl (ornithine), 2-aminoethyl(2,4-diaminobutyric acid), aminomethyl (2,3-diaminopropionic acid),3-guanidinopropan-1-yl (arginine). The L configuration is preferred ineach case.

Preference is given to compounds of the formula (I) in which

R^(A) is a group of the formula

in which

* means the point of linkage to the O atom,

L¹ is a bond,

L² is a bond or —CH₂—,

R¹ and R³ are independently of one another hydrogen or methyl,

R⁴ is hydrogen, methyl, propan-2-yl, propan-1-yl, 2-methylpropan-1-yl,1-methylpropan-1-yl, butan-1-yl, benzyl, p-hydroxybenzyl,imidazol-4-ylmethyl, hydroxymethyl, 1-hydroxyethyl, carbamoylmethyl or2-carbamoylethyl,

R⁶ is hydrogen, imidazol-4-ylmethyl, 4-aminobutan-1-yl,3-aminopropan-1-yl, 2-aminoethyl, aminomethyl or 3-guanidinopropan-1-yl,

L³ is straight-chain (C₂-C₄)-alkanediyl which is additionallysubstituted by amino,

R⁸ and R¹⁰ are independently of one another hydrogen or methyl,

m is the number 2, 3 or 4,

L⁴ is straight-chain (C₂-C₄)-alkanediyl which is additionallysubstituted by carboxyl,

R¹¹ is hydrogen or methyl, and

n is the number 2, 3 or 4,

and the salts, solvates and solvates of the salts thereof.

Particular preference is given to compounds of the formula (I) in which

R^(A) is a group of the formula

in which

* means the point of linkage to the O atom,

L¹ and L² are each a bond,

R⁴ is hydrogen, methyl, propan-2-yl, propan-1-yl, 2-methylpropan-1-yl,1-methylpropan-1-yl, butan-1-yl, benzyl, p-hydroxybenzyl,imidazol-4-ylmethyl, hydroxymethyl, 1-hydroxyethyl, carbamoylmethyl or2-carbamoylethyl,

R⁶ is imidazol-4-ylmethyl, 4-aminobutan-1-yl, 3-aminopropan-1-yl,2-aminoethyl, aminomethyl or 3-guanidinopropan-1-yl,

L³ is a group of the formula —CH(NH₂)—CH₂—, —CH₂—CH(NH₂)—,—CH₂—CH(NH₂)—CH₂—, —CH(NH₂)—CH₂—CH₂— or —CH₂—CH₂—CH(NH₂)—,

m is the number 2, 3 or 4,

L⁴ is a group of the formula **—CH₂—CH(COOH)— or **—CH₂—CH₂—CH(COOH)—,in which

** represents the point of linkage to the adjoining carbonyl group, and

n is the number 2 or 3,

and the salts, solvates and solvates of the salts thereof.

Very particular preference is given to compounds of the formula (I) inwhich

R^(A) is a group of the formula

in which

* means the point of linkage to the O atom,

L¹ and L² are each a bond,

R⁴ is hydrogen, methyl, propan-2-yl, 2-methylpropan-1-yl, benzyl,hydroxymethyl or 1-hydroxyethyl, and

R⁶ is imidazol-4-ylmethyl, 4-aminobutan-1-yl, 3-aminopropan-1-yl,2-aminoethyl, aminomethyl or 3-guanidinopropan-1-yl,

and the salts, solvates and solvates of the salts thereof.

Very particular preference is also given to compounds of the formula (I)in which

R^(A) is a group of the formula

in which

* means the point of linkage to the O atom,

L³ is a group of the formula —CH(NH₂)—CH₂—, —CH₂—CH(NH₂)—,—CH(NH₂)—CH₂—CH₂— or —CH₂—CH₂—CH(NH₂)—, and

m is the number 2 or 3,

and the salts, solvates and solvates of the salts thereof.

The invention further relates to a process for preparing the compoundsaccording to the invention of the formula (I), characterized in that thecompound (A)

either

[A] is esterified in an inert solvent in the presence of a condensingagent initially with a carboxylic acid of the formula (II), (III) or(IV)

in which L¹, L³, L⁴, R¹, R⁴, R⁵ and R¹¹ each have the meanings indicatedabove, and

PG¹ is a temporary amino protective group such as, for example,tert-butoxycarbonyl and

PG² is a temporary carboxyl protective group such as, for example,tert-butyl,

to give compounds of the formula (V), (VI) or (VII),

in which L¹, L³, L⁴, R¹, R⁴, R⁵, R¹¹, PG¹ and PG² each have the meaningsindicated above,

then, after elimination of the protective group PG¹ or PG², is coupledin an inert solvent in the presence of a condensing agent in the case ofcompound (V) with a compound of the formula (VIII), in the case ofcompound (VI) with a compound of the formula (IX) and in the case ofcompound (VII) with a compound of the formula (X)

in which L², R⁶, R⁷, R⁸, PG², m and n each have the meanings indicatedabove, and

R^(2a) and R^(3a), and R^(9a) and R^(10a), are in each case identical ordifferent and have the meanings of respectively R², R³, R⁹ and R¹⁰indicated above, or are a temporary amino protective group such as, forexample, tert-butoxycarbonyl,

and subsequently protective groups which are present where appropriateare removed again, or

[B] is coupled in an inert solvent in the presence of a condensing agentwith a compound of the formula (XI), (XII) or (XIII)

in which L¹, L², L³, L⁴, R¹, R⁴, R⁵, R⁶, R⁷, R⁸, R¹¹, m and n each havethe meanings indicated above,

R^(2a) and R^(3a), and R^(9a) and R^(10a), are in each case identical ordifferent and have the meanings of respectively R², R³, R⁹ and R¹⁰indicated above, or are a temporary amino protective group such as, forexample, tert-butoxycarbonyl, and

PG² is a temporary carboxyl protective group such as, for example,tert-butyl,

and subsequently protective groups which are present where appropriateare removed again,

and the resulting compounds of the formula (I) are converted whereappropriate with the appropriate (i) solvents and/or (ii) acids or basesinto the solvates, salts and/or solvates of the salts thereof.

The transformation (A)→(I) thus takes place either by sequentialcoupling of the individual carboxylic acid or amine components which aresuitably protected where appropriate (process variant [A]) or by directacylation with a suitably protected dipeptoid derivative (processvariant [B]). The coupling reactions (ester or amide formation) are inthis case carried out by known methods of peptide chemistry [cf., forexample, M. Bodanszky, Principles of Peptide Synthesis, Springer-Verlag,Berlin, 1993; H.-D. Jakubke and H. Jeschkeit, Aminosäuren, Peptide,Proteine, Verlag Chemie, Weinheim, 1982].

Examples of inert solvents for the coupling reactions are ethers such asdiethyl ether, tert-butyl methyl ether, dioxane, tetrahydrofuran, glycoldimethyl ether or diethylene glycol dimethyl ether, hydrocarbons such asbenzene, toluene, xylene, hexane, cyclohexane or petroleum fractions,halohydrocarbons such as dichloromethane, trichloromethane,tetrachloromethane, 1,2-dichloro-ethane, trichloroethylene orchlorobenzene, or other solvents such as acetone, ethyl acetate,pyridine, dimethyl sulfoxide, dimethylformamide,N,N′-dimethylpropyleneurea (DMPU), N-methylpyrrolidone (NMP) oracetonitrile. It is likewise possible to use mixtures of the solventsmentioned. Dichloromethane, dimethylformamide or mixtures of these twosolvents are preferred.

Examples of suitable condensing agents in these coupling reactions arecarbodiimides such as N,N′-diethyl-, N,N′-dipropyl-, N,N′-diisopropyl-,N,N′-dicyclohexylcarbodiimide (DCC) orN-(3-dimethylaminoisopropyl)-N′-ethylcarbodiimide hydrochloride (EDC),phosgene derivatives such as N,N′-carbonyldiimidazole (CD),1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2-oxazolium 3-sulfateor 2-tert-butyl-5-methylisoxazolium perchlorate, acylamino compoundssuch as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, or isobutylchloroformate, propanephosphonic anhydride, diethyl cyanophosphonate,bis-(2-oxo-3-oxazolidinyl)phosphoryl chloride,benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate,benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate(PyBOP), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TBTU),O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HBTU), 2-(2-oxo-1-(2H)-pyridyl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TPTU),O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) orO-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TCTU), where appropriate in combination with furtherauxiliaries such as 1-hydroxybenzotriazole (HOBt) orN-hydroxysuccinimide (HOSu), and as bases are alkali metal carbonates,e.g. sodium or potassium carbonate, or organic bases such astriethylamine, N-methylmorpholine, N-methylpiperidine,N,N-diisopropylethylamine or 4-N,N-dimethylaminopyridine.N-(3-Dimethylaminoisopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) incombination with 4-N,N-dimethylaminopyridine is preferably employed forester deformation. N-(3-Dimethylaminoisopropyl)-N′-ethylcarbodiimidehydrochloride (EDC) in combination with 1-hydroxybenzotriazole (HOBt) orN-hydroxysuccinimide (HOSu) and, where appropriate, a base such asN,N-diisopropylethylamine is preferably used for amide formation.

The couplings are generally carried out in a temperature range from 0°C. to +60° C., preferably at +20° C. to +40° C. The reactions can takeplace under normal, under elevated or under reduced pressure (e.g. from0.5 to 5 bar). They are generally carried out under atmosphericpressure.

The compounds of the formula (I) may also result directly in the form oftheir salts in the preparation by the processes described above. Thesesalts can be converted where appropriate by treatment with a base oracid in an inert solvent, by chromatographic methods or by ion exchangeresins, into the respective free bases or acids. Further salts of thecompounds according to the invention can also be prepared whereappropriate by exchange of counterions by means of ion exchangechromatography, for example with Amberlite® resins.

Functional groups which are present where appropriate in the radicalsR⁴, R⁶, L³ and/or L⁴—such as, in particular, amino, guanidine, hydroxy,mercapto and carboxyl groups—may, if expedient or necessary, also be intemporarily protected form in the reaction sequences described above.The introduction and removal of such protective groups takes place inthis connection by conventional methods known from peptide chemistry[see, for example, T.W. Greene and P.G.M. Wuts, Protective Groups inOrganic Synthesis, Wiley, New York, 1999; M. Bodanszky and A. Bodanszky,The Practice of Peptide Synthesis, Springer-Verlag, Berlin, 1984].

The amino and guanidine protective group which is preferably used istert-butoxycarbonyl (Boc) or benzyloxycarbonyl (Z). The protective grouppreferably employed for a hydroxy or carboxyl function is preferablytert-butyl or benzyl. Elimination of these protective groups is carriedout by conventional methods, preferably by reaction with a strong acidsuch as hydrogen chloride, hydrogen bromide or trifluoroacetic acid inan inert solvent such as dioxane, dichloromethane or acetic acid; theelimination can where appropriate also take place without an additionalinert solvent. In the case of benzyl and benzyloxycarbonyl as protectivegroup, these can also be removed by hydrogenolysis in the presence of apalladium catalyst. Elimination of the protective groups mentioned maywhere appropriate be carried out simultaneously in a one-pot reaction orin separate reaction steps.

The compounds of the formulae (II), (III), (IV), (VIII), (IX), (X),(XI), (XII) and (XIII) are commercially available, known from theliterature or can be prepared by methods customary in the literature.Thus, for example, compounds of the formulae (II) and (VIII) in which L¹or L² is —CH₂— can be obtained by known methods for chain extension ofcarboxylic acids, such as, for example, the Arndt-Eistert reactions[Eistert et al., Ber. Dtsch. Chem. Ges. 60, 1364-1370 (1927); Ye et al.,Chem. Rev. 94, 1091-1160 (1994); Cesar et al., Tetrahedron Lett. 42,7099-7102 (2001)] or the reaction with N-hydroxy-2-thiopyridone [cf.Barton et al., Tetrahedron Lett. 32, 3309-3312 (1991)], starting fromthe corresponding compounds in which L¹ or L² is a bond.

Preparation of compound (A),2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxyethoxy)phenyl]pyridine-3,5-dicarbonitrile,is described in WO 03/053441 as example 6.

The preparation of the compounds according to the invention can beillustrated by the following synthesis schemes:

The compounds according to the invention and their salts representuseful prodrugs of the active ingredient compound (A). On the one hand,they show good stability at various pH values and, on the other hand,they show efficient conversion into the active ingredient compound (A)at a physiological pH and in particular in vivo. The compounds accordingto the invention moreover have improved solubilities in aqueous or otherphysiologically tolerated media, making them suitable for therapeuticuse, in particular on intravenous administration. In addition, thebioavailability from suspension after oral administration is improved bycomparison with the parent substance (A).

The compounds of the formula (I) are suitable alone or in combinationwith one or more other active ingredients for the prophylaxis and/ortreatment of various disorders, for example and in particular disordersof the cardiovascular system (cardiovascular disorders), for cardioprotection following lesions of the heart, and of metabolic disorders.

Disorders of the cardiovascular system, or cardiovascular disorders,mean in the context of the present invention for example the followingdisorders: hypertension (high blood pressure), peripheral and cardiacvascular disorders, coronary heart disease, coronary restenosis such as,for example, restenosis following balloon dilatation of peripheral bloodvessels, myocardial infarction, acute coronary syndrome, acute coronarysyndrome with ST elevation, acute coronary syndrome without STelevation, stable and unstable angina pectoris, myocardialinsufficiency, princemetal angina, persistent ischemic dysfunction(“hibernating myocardium”), temporary postischemic dysfunction (“stunnedmyocardium”), heart failure, tachycardia, atrial tachycardia,arrhythmias, atrial and ventricular fibrillation, persistent atrialfibrillation, permanent atrial fibrillation, atrial fibrillation withnormal left ventricular function, atrial fibrillation with impaired leftventricular function, Wolff-Parkinson-White syndrome, disturbances ofperipheral blood flow, elevated levels of fibrinogen and of low densityLDL, and elevated concentrations of plasminogen activator inhibitor 1(PAI-1), especially hypertension, coronary heart disease, acute coronarysyndrome, angina pectoris, heart failure, myocardial infarction andatrial fibrillation.

In the context of the present invention, the term heart failure includesboth acute and chronic manifestations of heart failure, as well as morespecific or related types of disease, such as acute decompensated heartfailure, right heart failure, left heart failure, global failure,ischemic cardiomyopathy, dilated cardiomyopathy, congenital heartdefects, heart valve defects, heart failure associated with heart valvedefects, mitral stenosis, mitral insufficiency, aortic stenosis, aorticinsufficiency, tricuspid stenosis, tricuspid insufficiency, pulmonarystenosis, pulmonary valve insufficiency, combined heart valve defects,myocardial inflammation (myocarditis), chronic myocarditis, acutemyocarditis, viral myocarditis, diabetic heart failure, alcoholiccardiomyopathy, cardiac storage disorders, and diastolic and systolicheart failure.

The compounds according to the invention are further also suitable inparticular for reducing the area of myocardium affected by aninfarction, and for the prophylaxis of secondary infarctions.

The compounds according to the invention are furthermore suitable inparticular for the prophylaxis and/or treatment of thromboembolicdisorders, reperfusion damage following ischemia, micro- andmacromuscular lesions (vasculitis), arterial and venous thromboses,edemas, ischemias such as myocardial infarction, stroke and transientischemic attacks, for cardio protection in connection with coronaryartery bypass operations (CABG), primary percutaneous transluminalcoronary angioplasties (PTCAs), PTCAs after thrombolysis, rescue PTCA,heart transplants and open-heart operations, and for organ protection inconnection with transplants, bypass operations, catheter investigationsand other surgical procedures.

Further indication areas for which the compounds according to theinvention can be used are for example the prophylaxis and/or treatmentof disorders of the urogenital region, such as, for example, acute renalfailure, unstable bladder, urogenital incontinence, erectile dysfunctionand female sexual dysfunction, but also the prophylaxis and/or treatmentof inflammatory disorders such as, for example, inflammatory dermatosesand arthritis, especially rheumatoid arthritis, of disorders of thecentral nervous system and neurodegenerative impairments (post-strokeconditions, Alzheimer's disease, Parkinson's disease, dementia,Huntington's chorea, epilepsy, depression, multiple sclerosis), ofpainful conditions and migraine, hepatic fibrosis and cirrhosis of theliver, of cancers and of nausea and vomiting in connection with cancertherapies, and for wound healing.

A further indication area is for example the prophylaxis and/ortreatment of respiratory disorders such as, for example, asthma, chronicobstructive respiratory disorders (COPD, chronic bronchitis), pulmonaryemphysema, bronchiectasies, cystic fibrosis (mucoviscidosis) andpulmonary hypertension, especially pulmonary aterial hypertension.

Finally, the compounds according to the invention are also suitable forthe prophylaxis and/or treatment of metabolic disorders such as, forexample, diabetes, especially diabetes mellitus, gestational diabetes,insulin-dependent diabetes and non-insulin-dependent diabetes, diabeticsequelae such as, for example, retinopathy, nephropathy and neuropathy,metabolic disorders such as, for example, metabolic syndrome,hyperglycemia, hyperinsulinemia, insulin resistance, glucose intoleranceand obesity (adiposity), and arteriosclerosis and dyslipidemias(hypercholesterolemia, hypertriglyceridemia, elevated concentrations ofpost-prandial plasma triglycerides, hypoalphalipoproteinemia, combinedhyperlipidemias), especially, of diabetes, metabolic syndrome anddyslipidemias.

The present invention further relates to the use of the compoundsaccording to the invention for the treatment and/or prophylaxis ofdisorders, especially of the aforementioned disorders.

The present invention further relates to the use of the compoundsaccording to the invention for the manufacture of a medicament for thetreatment and/or prophylaxis of disorders, especially of theaforementioned disorders.

The present invention further relates to a method for the treatmentand/or prophylaxis of disorders, especially of the aforementioneddisorders, by using an effective amount of at least one of the compoundsaccording to the invention.

The compounds according to the invention can be employed alone or, ifrequired, in combination with other active ingredients. The presentinvention further relates to medicaments comprising at least one of thecompounds according to the invention and one or more further activeingredients, in particular for the treatment and/or prophylaxis of theaforementioned disorders.

Suitable combination active ingredients which may be mentioned by way ofexample and preferably are: lipid metabolism-altering activeingredients, antidiabetics, blood pressure-reducing agents, agents whichpromote blood flow and/or have antithrombotic effects, antiarrhythmics,antioxidants, chemokine receptor antagonists, p38 kinase inhibitors, NPYagonists, orexin agonists, anorectic agents, PAF-AH inhibitors,anti-inflammatory agents (COX inhibitors, LTB₄ receptor antagonists),and analgesics such as, for example, aspirin.

The present invention relates in particular to combinations of at leastone of the compounds according to the invention with at least one lipidmetabolism-altering active ingredient, antidiabetic, blood pressurereducing active ingredient, antiarrhythmic and/or agent havingantithrombotic effects.

The compounds according to the invention can preferably be combined withone or more

-   -   lipid metabolism-altering active ingredients, by way of example        and preferably from the group of HMG-CoA reductase inhibitors,        inhibitors of HMG-CoA reductase expression, squalene synthesis        inhibitors, ACAT inhibitors, LDL receptor inducers, cholesterol        absorption inhibitors, polymeric bile acid adsorbents, bile acid        reabsorption inhibitors, MTP inhibitors, lipase inhibitors, LPL        activators, fibrates, niacin, CETP inhibitors, PPAR-α, PPAR-γ        and/or PPAR-δ agonists, RXR modulators, FXR modulators, LXR        modulators, thyroid hormones and/or thyroid mimetics,        ATP-citrate lyase inhibitors, Lp(a) antagonists, cannabinoid        receptor 1 antagonists, leptin receptor agonists, bombesin        receptor agonists, histamine receptor agonists, and of        antioxidants/radical scavengers;    -   antidiabetics which are mentioned in the Rote Liste 2004/II,        Chapter 12, and, by way of example and preferably, those from        the group of sulfonylureas, biguanides, meglitinide derivatives,        glucosidase inhibitors, inhibitors of dipeptidyl-peptidase IV        (DPP-IV inhibitors), oxadiazolidinones, thiazolidinediones, GLP        1 receptor agonists, glucagon antagonists, insulin sensitizers,        CCK 1 receptor agonists, leptin receptor agonists, inhibitors of        hepatic enzymes involved in the stimulation of gluconeogenesis        and/or glycogenolysis, modulators of glucose uptake, and of        potassium channel openers such as, for example, those disclosed        in WO 97/26265 and WO 99/03861;    -   blood pressure-reducing active ingredients, by way of example        and preferably from the group of calcium antagonists,        angiotensin All antagonists, ACE inhibitors, rennin inhibitors,        beta-adrenoceptor antagonists, alpha-adrenoceptor antagonists,        diuretics, aldosterone antagonists, mineralocorticoid receptor        antagonists, ECE inhibitors, and of vasopeptidase inhibitors;    -   agents having antithrombotic effects, by way of example and        preferably from the group of platelet aggregation inhibitors or        of anticoagulants;    -   antiarrhythmics, especially those for the treatment of        supraventricular arrhythmias and tachycardias;    -   substances for the prophylaxis and treatment of ischemic and        reperfusion damage;    -   vasopressin receptor antagonists;    -   organic nitrates and NO donors;    -   compounds with positive inotropic activity;    -   compounds which inhibit the degradation of cyclic guanosine        monophosphate (cGMP) and/or cyclic adenosine monophosphate        (cAMP), such as, for example, inhibitors of phosphodiesterases        (PDE) 1, 2, 3, 4 and/or 5, especially PDE 5 inhibitors such as        sildenafil, vardenafil and tadalafil, and PDE 3 inhibitors such        as milrinone;    -   natriuretic peptides such as, for example, atrial natriuretic        peptide (ANP, anaritide), B-type natriuretic peptide or brain        natriuretic peptide (BNP, nesiritide), C-type natriuretic        peptide (CNP) and urodilatin;    -   agonists of the prostacyclin receptor (IP receptor), such as,        for example iloprost, beraprost and cicaprost;    -   calcium sensitizers such as by way of example and preferably        levosimendan;    -   potassium supplements;    -   NO and heme-independent activators of guanylate cyclase, such as        in particular the compounds described in WO 01/19355, WO        01/19776, WO 01/19778, WO 01/19780, WO 02/070462 and WO        02/070510;    -   NO-independent but heme-dependent stimulators of guanylate        cyclase, such as in particular the compounds described in WO        00/06568, WO 00/06569, WO 02/42301 and WO 03/095451;    -   Inhibitors of human neutrophil elastase (HNE), such as, for        example, sivelestat and DX-890 (reltran);    -   compounds which inhibit the signal transduction cascade, such        as, for example, tyrosine kinase inhibitors, especially        sorafenib, imatinib, gefitinib and erlotinib;    -   compounds which influence the energy metabolism of the heart,        such as, for example, etomoxir, dichloroacetate, ranolazine and        trimetazidine;    -   analgesics; and/or    -   substances for the prophylaxis and treatment of nausea and        vomiting

Lipid metabolism-altering active ingredients preferably mean compoundsfrom the group of HMG-CoA reductase inhibitors, squalene synthesisinhibitors, ACAT inhibitors, choleseterol absorption inhibitors, MTPinhibitors, lipase inhibitors, thyroid hormones and/or thyroid mimetics,niacin receptor agonists, CETP inhibitors, PPAR-α agonists, PPAR-γagonists, PPAR-δ agonists, polymeric bile acid adsorbents, bile acidreabsorption inhibitors, antioxidants/radical scavengers, andcannabinoid receptor 1 antagonists.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with an HMG-CoA reductaseinhibitor from the class of statins, such as by way of example andpreferably lovastatin, simvastatin, pravastatin, fluvastatin,atorvastatin, rosuvastatin, cerivastatin or pitavastatin.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a squalene synthesisinhibitor, such as by way of example and preferably BMS-188494 orTAK-475.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with an ACAT inhibitor,such as by way of example and preferably avasimibe, melinamide,pactimibe, eflucimibe or SMP-797.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a cholesterolabsorption inhibitor, such as by way of example and preferablyezetimibe, tiqueside or pamaqueside.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with an MTP inhibitor,such as by way of example and preferably implitapide, BMS-201038,R-103757 or JTT-130.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a lipase inhibitor,such as by way of example and preferably orlistat.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a thyroid hormoneand/or thyroid mimetic, such as by way of example and preferablyD-thyroxine or 3,5,3′-triiodothyronine (T3).

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with an agonist of theniacin receptor, such as by way of example and preferably niacin,acipimox, acifran or radecol.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a CETP inhibitor,such as by way of example and preferably torcetrapib, JTT-705, BAY60-5521, BAY 78-7499 or CETP vaccine (Avant).

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a PPAR-γ agonist,such as by way of example and preferably pioglitazone or rosiglitazone.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a PPAR-δ agonist,such as by way of example and preferably GW-501516 or BAY 68-5042.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a polymeric bile acidadsorbent, such as by way of example and preferably cholestyramine,colestipol, colesolvam, CholestaGel or colestimide.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a bile acidreabsorption inhibitor, such as by way of example and preferably ASBT(=IBAT) inhibitors, such as, for example, AZD-7806, S-8921, AK-105,BARI-1741, SC-435 or SC-635.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with anantioxidant/radical scavenger, such as by way of example and preferablyprobucol, AGI-1067, BO-653 or AEOL-10150.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a cannabinoidreceptor 1 antagonist, such as by way of example and preferablyrimonabant or SR-147778.

Antidiabetics preferably mean insulin and insulin derivatives, andorally active hypoglycemic active ingredients. Insulin and insulinderivatives includes in this connection both insulins of animal, humanor biotechnological origin and mixtures thereof. The orally activehypoglycemic active ingredients preferably include sulfonylureas,biguanides, meglitinide derivatives, glucosidase inhibitors, DPP-IVinhibitors and PPAR-γ agonists.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with insulin.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a sulfonylurea, suchas by way of example and preferably tolbutamide, glibenclamide,glimepiride, glipizide or gliclazide.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a biguanide, such asby way of example and preferably metformin.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a meglitinidederivative, such as by way of example and preferably repaglinide ornateglinide.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a glucosidaseinhibitor, such as by way of example and preferably miglitol oracarbose.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a DPP-IV inhibitor,such as by way of example and preferably sitagliptin or vildagliptin.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a PPAR-γ agonist, forexample from the class of thiazolidinediones, such as by way of exampleand preferably pioglitazone or rosiglitazone.

Blood pressure-reducing agents preferably mean compounds from the groupof calcium antagonists, angiotensin AII antagonists, ACE inhibitors,renin inhibitors, beta-adrenoceptor antagonists, alpha-adrenoceptorantagonists and diuretics.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a calcium antagonist,such as by way of example and preferably nifedipine, amlodipine,verapamil or diltiazem.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with an angiotensin Allantagonist, such as by way of example and preferably losartan,valsartan, candesartan, embusartan, olmesartan or telmisartan.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with an ACE inhibitor,such as by way of example and preferably enalapril, captopril,lisinopril, ramipril, delapril, fosinopril, quinopril, perindopril ortrandopril.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a renin inhibitor,such as by way of example and preferably aliskiren, SPP-600 or SPP-800.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a beta-adrenoceptorantagonist, such as by way of example and preferably propranolol,atenolol, timolol, pindolol, alprenolol, oxprenolol, penbutolol,bupranolol, metipranolol, nadolol, mepindolol, carazalol, sotalol,metoprolol, betaxolol, celiprolol, bisoprolol, carteolol, esmolol,labetalol, carvedilol, adaprolol, landiolol, nebivolol, epanolol orbucindolol.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with an alpha-adrenoceptorantagonist, such as by way of example and preferably prazosin.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a diuretic, such asby way of example and preferably furosemide, bumetanide, torsemide,bendroflumethiazide, chlorthiazide, hydrochlorthiazide,hydroflumethiazide, methyclothiazide, polythiazide, trichlormethiazide,chlorthalidone, indapamide, metolazone, quinethazone, acetazolamide,dichlorphenamide, methazolamide, glycerol, isosorbide, mannitol,amiloride or triamterene.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with an aldosterone ormineralocorticoid receptor antagonist, such as by way of example andpreferably spironolactone or eplerenone.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a vasopressinreceptor antagonist, such as by way of example and preferablyconivaptan, tolvaptan, lixivaptan or SR-121463.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with an organic nitrate orNO donor, such as by way of example and preferably sodium nitroprusside,glycerol nitrate, isosorbide mononitrate, isosorbide dinitrate,molsidomine or SIN-1, or in combination with inhaled NO.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a compound havingpositive inotropic activity, such as by way of example and preferablycardiac glycosides (digoxin) and beta-adrenergic and dopaminergicagonists such as isoproterenol, adrenaline, noradrenaline, dopamine ordobutamine.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with antisympathotonicssuch as reserpine, clonidine or alpha-methyldopa, or in combination withpotassium channel agonists such as minoxidil, diazoxide, dihydralazineor hydralazine.

Agents having an antithrombotic effect preferably mean compounds fromthe group of platelet aggregation inhibitors or of anticoagulants.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a plateletaggregation inhibitor, such as by way of example and preferably aspirin,clopidogrel, ticlopidine or dipyridamole.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a thrombin inhibitor,such as by way of example and preferably ximelagatran, melagatran,bivalirudin or clexane.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a GPIIb/IIIaantagonist, such as by way of example and preferably tirofiban orabciximab.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a factor Xainhibitor, such as by way of example and preferably rivaroxaban (BAY59-7939), DU-176b, apixaban, otamixaban, fidexaban, razaxaban,fondaparinux, idraparinux, PMD-3112, YM-150, KFA-1982, EMD-503982,MCM-17, MLN-1021, DX 9065a, DPC 906, JTV 803, SSR-126512 or SSR-128428.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with heparin or a lowmolecular weight (LMW) heparin derivative.

In a preferred embodiment of the invention, the compounds according tothe invention are administered in combination with a vitamin Kantagonist, such as by way of example and preferably coumarin.

Antiarrhythmics preferably means substances from the group of class laantiarrhythmics (e.g. quinidine), of class Ic antiarrhythmics (e.g.flecainide, propafenone), of class II antiarrhythmics (e.g. metoprolol,atenolol, sotalol, oxprenolol and other beta-receptor blockers), ofclass III antiarrhythmics (e.g. sotalol, amiodarone) and of class IVantiarrhythmics (e.g. digoxin, and verapamil, diltiazem and othercalcium antagonists).

Particular preference is given in the context of the present inventionto combinations comprising at least one of the compounds according tothe invention and one or more further active ingredients selected fromthe group consisting of HMG-CoA reductase inhibitors (statins),diuretics, beta-adrenoceptor antagonists, alpha-adrenoceptorantagonists, organic nitrates and NO donors, calcium antagonists, ACEinhibitors, angiotensin All antagonists, aldosterone andmineralocorticoid receptor antagonists, vasopressin receptorantagonists, platelet aggregation inhibitors, anticoagulants andantiarrhythmics, and to the use thereof for the treatment and/orprophylaxis of the aforementioned disorders.

The present invention further relates to medicaments which comprise atleast one compound according to the invention, normally together withone or more inert, non-toxic, pharmaceutically suitable excipients, andto the use thereof for the aforementioned purposes.

The compounds according to the invention can act systemically and/orlocally. For this purpose, they can be administered in a suitable waysuch as, for example, by the oral, parenteral, pulmonary, nasal,sublingual, lingual, buccal, rectal, dermal, transdermal, conjunctivalor otic route or as an implant or stent. The compounds according to theinvention can be administered in administration forms suitable for theseadministration routes.

Suitable for oral administration are administration forms which functionaccording to the prior art and deliver the compounds according to theinvention rapidly and/or in modified fashion, and which contain thecompounds according to the invention in crystalline and/or amorphizedand/or dissolved form, such as, for example, tablets (uncoated or coatedtablets, for example having enteric coatings or coatings which areinsoluble or dissolve with a delay and control the release of thecompound according to the invention), tablets which disintegrate rapidlyin the mouth, or films/wafers, films/lyophilizates, capsules (forexample hard or soft gelatin capsules), sugar-coated tablets, granules,pellets, powders, emulsions, suspensions, aerosols or solutions.

Parenteral administration can take place with avoidance of an absorptionstep (e.g. intravenous, intraarterial, intracardiac, intraspinal orintralumbar) or with inclusion of an absorption (e.g. intramuscular,subcutaneous, intracutaneous, percutaneous or intraperitoneal).Administration forms suitable for parenteral administration are, interalia, preparations for injection and infusion in the form of solutions,suspensions, emulsions, lyophilizates or sterile powders.

Suitable for the other administration routes are, for example,pharmaceutical forms for inhalation (inter alia powder inhalers,nebulizers), nasal drops, solutions or sprays, tablets for lingual,sublingual or buccal administration, films/wafers or capsules,suppositories, preparations for the eyes or ears, vaginal capsules,aqueous suspensions (lotions, shaking mixtures), lipophilic suspensions,ointments, creams, transdermal therapeutic systems (such as, forexample, patches), milk, pastes, foams, dusting powders, implants orstents.

Oral or parenteral administration is preferred, especially oral andintravenous administration.

The compounds according to the invention can be converted into thestated administration forms. This can take place in a manner known perse by mixing with inert, non-toxic, pharmaceutically suitableexcipients. These excipients include, inter alia, carriers (for examplemicrocrystalline cellulose, lactose, mannitol), solvents (e.g. liquidpolyethylene glycols), emulsifiers and dispersants or wetting agents(for example sodium dodecyl sulfate, polyoxy-sorbitan oleate), binders(for example polyvinylpyrrolidone), synthetic and natural polymers (forexample albumin), stabilizers (e.g. antioxidants such as, for example,ascorbic acid), colorants (e.g. inorganic pigments such as, for example,iron oxides) and masking flavors and/or odors.

It has generally proved advantageous to administer on parenteraladministration amounts of about 0.001 to 1 mg/kg, preferably about 0.01to 0.5 mg/kg, of body weight to achieve effective results, and on oraladministration the dosage is about 0.01 to 100 mg/kg, preferably about0.01 to 20 mg/kg, and very particularly preferably 0.1 to 10 mg/kg, ofbody weight.

It may nevertheless be necessary where appropriate to deviate from thestated amounts, in particular as a function of the body weight, route ofadministration, individual response to the active ingredient, nature ofthe preparation and time or interval over which administration takesplace. Thus, it may be sufficient in some cases to make do with lessthan the aforementioned minimum amount, whereas in other cases thestated upper limit must be exceeded. It may in the event ofadministration of larger amounts be advisable to divide these into aplurality of individual doses over the day.

The following exemplary embodiments illustrate the invention. Theinvention is not restricted to the examples.

The percentage data in the following tests and examples are, unlessindicated otherwise, percentages by weight; parts are parts by weight.Solvent ratios, dilution ratios and concentration data for theliquid/liquid solutions are in each case based on volume.

A. EXAMPLES

Abbreviations and Acronyms:

Boc Tert-Butoxycarbonyl

DIEA N,N-Diisopropylethylamine

DMAP 4-N,N-Dimethylaminopyridine

DMF N,N-Dimethylformamide

DMSO Dimethyl sulfoxide

EDC 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

ESI Electrospray ionization (in MS)

h Hour(s)

HOBt 1-Hydroxybenzotriazole

HPLC High pressure, high performance liquid chromatography

LC-MS Coupled liquid chromatography-mass spectrometry

min Minute(s)

MS Mass spectrometry

NMR Nuclear magnetic resonance spectrometry

p para

Pd/C Palladium on activated carbon

Ph Phenyl

quant. quantitative (for yield)

RT Room temperature

R_(t) Retention time (in HPLC)

tert. Tertiary

TFA Trifluoroacetic acid

THF Tetrahydrofuran

UV Ultraviolet spectrometry

v/v Volume to volume ratio (of a solution)

Z Benzyloxycarbonyl

LC-MS and HPLC Methods:

Method 1 (LC-MS):

MS instrument type: Micromass ZQ; HPLC instrument type: Waters Alliance2795; column: Phenomenex Synergi 2μ Hydro-RP Mercury 20 mm×4 mm; eluentA: 1 l water+0.5 ml 50% formic acid, eluent B: 1 l acetonitrile+0.5 ml50% formic acid; gradient: 0.0 min 90% A→2.5 min 30% A→3.0 min 5% A→4.5min 5% A; flow rate: 0.0 min 1 ml/min→2.5 min/3.0 min/4.5 min 2 ml/min;oven: 50° C.; UV detection: 210 nm.

Method 2 (LC-MS):

Instrument: Micromass Quattro LCZ with HPLC Agilent Series 1100; column:Phenomenex Synergi 2μ Hydro-RP Mercury 20 mm×4 mm; eluent A: 1 lwater+0.5 ml 50% formic acid, eluent B: 1 l acetonitrile+0.5 ml 50%formic acid; gradient: 0.0 min 90% A→2.5 min 30% A→3.0 min 5% A→4.5 min5% A; flow rate: 0.0 min 1 ml/min→2.5 min/3.0 min/4.5 min 2 ml/min;oven: 50° C.; UV detection: 208-400 nm.

Method 3 (LC-MS):

MS instrument type: Micromass ZQ; HPLC instrument type: HP 1100 Series;UV DAD; column: Phenomenex Synergi 2μ Hydro-RP Mercury 20 mm×4 mm;eluent A: 1 l water+0.5 ml 50% formic acid, eluent B: 1 lacetonitrile+0.5 ml 50% formic acid; gradient: 0.0 min 90% A→2.5 min 30%A→3.0 min 5% A→4.5 min 5% A; flow rate: 0.0 min 1 ml/min→2.5 min/3.0min/4.5 min 2 ml/min; oven: 50° C.; UV detection: 210 nm.

Method 4 (LC-MS):

MS instrument type: Micromass ZQ; HPLC instrument type: HP 1100 Series;UV DAD; column: Phenomenex Gemini 3μ 30 mm×3.00 mm; eluent A: 1 lwater+0.5 ml 50% formic acid, eluent B: 1 l acetonitrile+0.5 ml 50%formic acid; gradient: 0.0 min 90% A→2.5 min 30% A→3.0 min 5% A→4.5 min5% A; flow rate: 0.0 min 1 ml/min→2.5 min/3.0 min/4.5 min 2 ml/min;oven: 50° C.; UV detection: 210 nm.

Method 5 (Preparative HPLC):

HPLC instrument type: Abimed/Gilson Pump 305/306; Manometric Module 806;UV Knauer Variable Wavelength Monitor; column: Gromsil C18, 10 nm, 250mm×30 mm; eluent A: 1 l water+0.5 ml 99% trifluoroacetic acid, eluent B:1 l acetonitrile; gradient: 0.0 min 2% B→10 min 2% B→50 min 90% B; flowrate: 20 ml/min; volume: 628 ml A and 372 ml B.

Method 6a (Preparative HPLC):

Column: VP 250/21 Nukleodur 100-5 C18 ec, Macherey & Nagel Nr. 762002;eluent A: water/0.01% trifluoroacetic acid, eluent B: acetonitrile/0.01%trifluoroacetic acid; gradient: 0 min 0% B→20 min 20% B→40 min 20% B→60min 30% B→80 min 30% B→90 min 100% B→132 min 100% B; flow rate: 5ml/min; temperature: RT; UV detection: 210 nm.

Method 6b (Preparative HPLC):

Column: VP 250/21 Nukleodur 100-5 C18 ec, Macherey & Nagel Nr. 762002;eluent A: 1 liter water/1 ml 99% trifluoroacetic acid, eluent B: 1 literacetonitrile/1 ml 99% trifluoroacetic acid; gradient: 0 min 30% B→20 min50% B→40 min 80% B→60 min 100% B; flow rate: 5 ml/min; temperature: RT;UV detection: 210 nm.

Method 7 (Analytical HPLC):

Column: XTerra 3.9×150 WAT 186000478; eluent A: 10 ml 70% perchloricacid in 2.5 liters water, eluent B: acetonitrile; gradient: 0.0 min 20%B→1 min 20% B→4 min 90% B→9 min 90% B; temperature: RT; flow rate: 1ml/min.

Method 8 (LC-MS):

Instrument: Micromass Quattro LCZ with HPLC Agilent Series 1100; column:Phenomenex Onyx Monolithic C18, 100 mm×3 mm; eluent A: 1 l water+0.5 ml50% formic acid, eluent B: 1 l acetonitrile+0.5 ml 50% formic acid;gradient: 0.0 min 90% A→2 min 65% A→4.5 min 5% A→6 min 5% A; flow rate:2 ml/min; oven: 40° C.; UV detection: 208-400 nm.

Method 9 (LC-MS):

Instrument: Micromass Platform LCZ with HPLC Agilent Series 1100;column: Thermo Hypersil GOLD 3μ, 20 mm×4 mm; eluent A: 1 l water+0.5 ml50% formic acid, eluent B: 1 l acetonitrile+0.5 ml 50% formic acid;gradient: 0.0 min 100% A→0.2 min 100% A→2.9 min 30% A→3.1 min 10% A→5.5min 10% A; oven: 50° C.; flow rate: 0.8 ml/min; UV detection: 210 nm.

Method 10 (LC-MS):

MS instrument type: Micromass ZQ; HPLC instrument type: HP 1100 Series;UV DAD; column: Phenomenex Gemini 3μ 30 mm×3.00 mm; eluent A: 1 lwater+0.5 ml 50% formic acid, eluent B: 1 l acetonitrile+0.5 ml 50%formic acid; gradient: 0.0 min 90% A→2.5 min 30% A→3.0 min 5% A→4.5 min5% A; flow rate: 0.0 min 1 ml/min→2.5 min/3.0 min/4.5 min 2 ml/min;oven: 50° C.; UV detection: 210 nm.

Method 11 (LC-MS):

MS instrument type: Micromass ZQ; HPLC instrument type: Waters Alliance2795; column: Phenomenex Synergi 2.5μ MAX-RP 100A Mercury 20 mm×4 mm;eluent A: 1 l water+0.5 ml 50% formic acid, eluent B: 1 lacetonitrile+0.5 ml 50% formic acid; gradient: 0.0 min 90% A→0.1 min 90%A→3.0 min 5% A→4.0 min 5% A→4.01 min 90% A; flow rate: 2 ml/min; oven:50° C.; UV detection: 210 nm.

Method 12 (LC-MS):

Instrument: Micromass Quattro LCZ with HPLC Agilent Series 1100; column:Phenomenex Synergi 2.5μ MAX-RP 100A Mercury 20 mm×4 mm; eluent A: 1 lwater+0.5 ml 50% formic acid, eluent B: 1 l acetonitrile+0.5 ml 50%formic acid; gradient: 0.0 min 90% A→0.1 min 90% A→3.0 min 5% A→4.0 min5% A→4.1 min 90% A; flow rate: 2 ml/min; oven: 50° C.; UV detection:208-400 nm.

Method 13 (LC-MS):

Instrument: Micromass QuattroPremier with Waters UPLC Acquity; column:Thermo Hypersil GOLD 1.9μ, 50 mm×1 mm; eluent A: 1 l water+0.5 ml 50%formic acid, eluent B: 1 l acetonitrile+0.5 ml 50% formic acid;gradient: 0.0 min 100% A→0.1 min 100% A→1.5 min 10% A→2.2 min 10% A;oven: 50° C.; flow rate: 0.33 ml/min; UV detection: 210 nm.

Starting Compounds and Intermediates:

Example 1A2-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylL-valinate trifluoroacetate

1 g (1.92 mmol) of2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxy-ethoxy)phenyl]pyridine-3,5-dicarbonitrile[WO 03/053441, example 6], 0.460 g (2.11 mmol) of N-Boc-L-valine, 0.442g (2.31 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride and 0.023 g (0.19 mmol) of 4-dimethylaminopyridine aremixed in 40 ml of dichloromethane and 10 ml of DMF and stirred at roomtemperature overnight. A clear solution results. The mixture is thenpoured into a mixture of half-saturated ammonium chloride solution anddichloromethane. The organic phase is separated off, washed successivelywith water, saturated sodium bicarbonate solution and saturated sodiumchloride solution, dried over magnesium sulfate, filtered andconcentrated. The residue is purified by flash chromatography on silicagel with dichloromethane/ethyl acetate as eluent (gradient10:1→7:1→5:1). The appropriate fractions are combined and the solvent isremoved in vacuo. After the residue has been dried under high vacuum,0.85 g (62% of theory) of the Boc-protected intermediate remains.

The residue is taken up in 5 ml of dichloromethane and 5 ml of anhydroustrifluoroacetic acid, and the solution is stirred at room temperaturefor 2 h. The mixture is then concentrated to dryness, and the residue isstirred with ethyl acetate. The precipitate which separates out isfiltered off, washed with diethyl ether and then dried under highvacuum. 935 mg (quant.) of the title compound result as colorlesscrystals.

HPLC (Method 7): R_(t)=5.5 min;

LC-MS (Method 10): R_(t)=2.06 min; MS (ESIpos): m/z=619 (M′H)⁺.

Example 2A2-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylL-alaninate trifluoroacetate

1.5 g (2.88 mmol) of2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxyethoxy)phenyl]pyridine-3,5-dicarbonitrile,1.64 g (8.66 mmol) of N-Boc-L-alanine, 0.719 g (3.75 mmol) of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.176 g(1.44 mmol) of 4-dimethylaminopyridine are mixed in 25 ml ofdichloromethane and 25 ml of DMF and stirred at room temperature for 2h. The mixture is then concentrated in vacuo and the residue is taken upin ethyl acetate. The solution is extracted twice with 5% strengthcitric acid and twice with sodium bicarbonate solution. The organicphase is concentrated and the residue is stirred with 50 ml of diethylether and 50 ml of pentane. The precipitate is filtered off with suctionand washed with pentane. After the residue has been dried under highvacuum, 1.23 g (62% of theory) of the Boc-protected intermediate remain.

The residue is taken up in 18 ml of dichloromethane and 2 ml ofanhydrous trifluoroacetic acid, and the solution is treated in anultrasonic bath at room temperature for 1 h. The mixture is thenconcentrated to dryness, and the remaining residue is stirred withdiethyl ether. The precipitate which separates out is filtered off,washed with diethyl ether and then dried under high vacuum. 1200 mg (96%of theory) of the title compound result as colorless crystals.

HPLC (Method 7): R_(t)=5.3 min;

LC-MS (Method 12): R_(t)=1.73 min; MS (ESIpos): m/z=591 (M+H)⁺.

Example 3A2-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylO-tert-butyl-L-serinate

1 g (1.92 mmol) of2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxy-ethoxy)phenyl]pyridine-3,5-dicarbonitrile,0.612 g (2.12 mmol) of N-(tert-butoxycarbonyl)-O-tert-L-serine, 0.442 g(2.31 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride and 0.024 g (0.192 mmol) of 4-dimethylaminopyridine aremixed in 40 ml of dichloromethane and 10 ml of DMF and stirred at roomtemperature overnight. The mixture is then poured into a mixture ofhalf-saturated ammonium chloride solution and dichloromethane. Theorganic phase is separated off, washed successively with water,saturated sodium bicarbonate solution and saturated sodium chloridesolution, dried over magnesium sulfate, filtered and concentrated. Theresidue is taken up in dichloromethane and mixed with diethyl ether. Theprecipitate which separates out is filtered off with suction and washedwith diethyl ether. After drying under high vacuum, 1.25 g (85% oftheory) of the protected intermediate remain.

The residue is taken up in 100 ml of dichloromethane and 10 ml ofanhydrous trifluoroacetic acid, and the solution is stirred at roomtemperature for 1 h. The mixture is then poured into a mixture ofhalf-saturated sodium bicarbonate solution and dichloromethane. Theorganic phase is separated off, dried over magnesium sulfate, filteredand concentrated. Drying under high vacuum results in 1020 mg (95% oftheory) of the title compound as a colorless powder.

HPLC (Method 7): R_(t)=5.4 min;

LC-MS (Method 11): R_(t)=1.65 min; MS (ESIpos): m/z=663 (M+H)⁺.

Example 4AN²-(tert.-Butoxycarbonyl)-N-{2-[(tert-butoxycarbonyl)amino]ethyl}-L-α-glutamine

1.5 g (4.45 mmol) of(2S)-5-(benzyloxy)-2-[(tert-butoxycarbonyl)amino]-5-oxopentanoic acid,783 mg (4.89 mmol) of tert-butyl (2-aminoethyl)carbamate, 938 mg (4.89mmol) of 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochlorideand 749 mg (0.489 mmol) of 1-hydroxy-1H-benzotriazole hydrate are mixedin 140 ml of DMF and stirred at room temperature overnight. The mixtureis then poured into a mixture of half-saturated ammonium chloridesolution and ethyl acetate. The organic phase is separated off, washedsuccessively with water, saturated sodium bicarbonate solution andsaturated sodium chloride solution, dried over magnesium sulfate,filtered and concentrated. The residue is stirred with diethyl ether.The precipitate which separates out is filtered off with suction, washedwith diethyl ether and dried under high vacuum. 1.9 g (89% of theory) ofthe protected intermediate remain.

LC-MS (Method 12): R_(t)=2.19 min; MS (ESIpos): m/z=480 (M+H)⁺.

1.9 g (3.96 mmol) of the resulting intermediate are dissolved in 125 mlof methanol and, after addition of 250 mg of 10% palladium on activatedcarbon, hydrogenated under atmospheric pressure at RT for 2 h. Thecatalyst is then filtered off, and the solvent is removed in vacuo. 1500mg (97% of theory) of the title compound are obtained as a colorlessfoam.

LC-MS (Method 10): R_(t)=1.94 min; MS (ESIpos): m/z=390 (M+H)⁺.

Example 5AN²-(tert-Butoxycarbonyl)-N-{2-[(tert-butoxycarbonyl)amino]ethyl}-L-glutamine

1.5 g (4.45 mmol) of(4S)-5-(benzyloxy)-2-[(tert-butoxycarbonyl)amino]-5-oxopentanoic acid,783 mg (4.89 mmol) of tert-butyl (2-aminoethyl)carbamate, 938 mg (4.89mmol) of 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochlorideand 749 mg (0.489 mmol) of 1-hydroxy-1H-benzotriazole hydrate are mixedin 140 ml of DMF and stirred at room temperature overnight. The mixtureis then poured into a mixture of half-saturated ammonium chloridesolution and ethyl acetate. The organic phase is separated off, washedsuccessively with water, saturated sodium bicarbonate solution andsaturated sodium chloride solution, dried over magnesium sulfate,filtered and concentrated. The residue is stirred with diethyl ether.The precipitate which separates out is filtered off with suction, washedwith diethyl ether and dried under high vacuum. 2.1 g (98% of theory) ofthe protected intermediate remain.

LC-MS (Method 10): R_(t)=2.47 min; MS (ESIpos): m/z=480 (M+H)⁺.

2.1 g (4.38 mmol) of the resulting intermediate are dissolved in 140 mlof methanol and, after addition of 250 mg of 10% palladium on activatedcarbon, hydrogenated under atmospheric pressure at RT for 2 h. Thecatalyst is then filtered off and the solvent is removed in vacuo. 1540mg (90% of theory) of the title compound are obtained as a colorlessfoam.

LC-MS (Method 11): R_(t)=1.35 min; MS (ESIpos): m/z=390 (M+H)⁺.

Example 6AN²-(tert-Butoxycarbonyl)-N-{2-[(tert.-butoxycarbonyl)amino]ethyl}-L-asparagine

1.5 g (4.64 mmol) of(3S)-4-(benzyloxy)-3-[(tert-butoxycarbonyl)amino]-4-oxobutyric acid, 818mg (5.1 mmol) of tert-butyl-(2-aminoethyl)carbamate, 978 mg (5.1 mmol)of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 781mg (5.1 mmol) of 1-hydroxy-1H-benzotriazole hydrate are mixed in 75 mlof DMF and stirred at room temperature overnight. The mixture is thenpoured into a mixture of half-saturated ammonium chloride solution andethyl acetate. The organic phase is separated off, washed successivelywith water, saturated sodium bicarbonate solution and saturated sodiumchloride solution, dried over magnesium sulfate, filtered andconcentrated. The residue is stirred with diethyl ether. The precipitatewhich separates out is filtered off with suction, washed with diethylether and dried under high vacuum. 2.1 g (81% of theory) of theprotected intermediate remain.

LC-MS (Method 10): R_(t)=2.47 min; MS (ESIpos): m/z=466 (M+H)⁺.

2.1 g (4.51 mmol) of the resulting intermediate are dissolved in 140 mlof methanol and, after addition of 250 mg of 10% palladium on activatedcarbon, hydrogenated under atmospheric pressure at RT for 2 h. Thecatalyst is then filtered off and the solvent is removed in vacuo. 1690mg (99% of theory) of the title compound are obtained as a colorlessfoam.

LC-MS (Method 11): R_(t)=1.35 min; MS (ESIpos): m/z=376 (M+H)⁺.

Example 7A5-(2-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-3,5-dicyanopyridin-4-yl]-phenoxy}ethyl)1-tert-butyl L-glutamate

3.117 g (6 mmol) of2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxyethoxy)phenyl]pyridine-3,5-dicarbonitrile,2 g (6.59 mmol) of(4S)-5-tert-butoxy-4-[(tert-butoxycarbonyl)amino]-5-oxopentanoate, 1.38g (7.19 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride and 0.073 g (0.6 mmol) of 4-dimethylaminopyridine aremixed in 80 ml of dichloromethane and 20 ml of DMF and stirred at roomtemperature overnight. The mixture is then poured into a mixture ofhalf-saturated ammonium chloride solution and dichloromethane. Theorganic phase is separated off, washed successively with water,saturated sodium bicarbonate solution and saturated sodium chloridesolution, dried over magnesium sulfate, filtered and concentrated. Theresidue is precipitated from dichloromethane with petroleum ether. Theproduct is filtered off with suction, washed with diethyl ether anddried under high vacuum. 4.44 g (92% of theory) of the protectedintermediate remain.

LC-MS (Method 10): R_(t)=3.38 min; MS (ESIpos): m/z=805 (M+H)⁺.

57 mg (0.07 mmol) of the resulting intermediate are taken up in 6 ml ofdichloromethane and 0.6 ml of anhydrous trifluoroacetic acid, and thesolution is stirred at room temperature for 2.5 h. The mixture is thenpoured into a mixture of half-saturated sodium bicarbonate solution anddichloromethane. The organic phase is separated off, dried overmagnesium sulfate, filtered and concentrated. Drying under high vacuumresults in 50 mg (quant.) of the title compound as a colorless powder.

HPLC (Method 7): R_(t)=5.4 min;

LC-MS (Method 11): R_(t)=1.72 min; MS (ESIpos): m/z=705 (M+H)⁺.

Example 8A2-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}sulfanyl)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylL-leucinate trifluoroacetate

The title compound is prepared in analogy to example 1A starting from2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxyethoxy)phenyl]pyridine-3,5-dicarbonitrileand Boc-L-leucine.

HPLC (Method 7): R_(t)=5.5 min;

LC-MS (Method 12): R_(t)=1.75 min; MS (ESIpos): m/z=633 (M+H)⁺.

Example 9A2-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}sulfanyl)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylD-alaninate trifluoroacetate

The title compound is prepared in analogy to example 2A starting from2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxyethoxy)phenyl]pyridine-3,5-dicarbonitrileand Boc-D-alanine.

HPLC (Method 7): R_(t)=5.2 min;

LC-MS (Method 13): R_(t)=1.15 min; MS (ESIpos): m/z=591 (M+H)⁺.

Example 10AN²-(tert-Butoxycarbonyl)-N-{2-[(tert-butoxycarbonyl)amino]ethyl}-D-α-glutamine

The title compound is prepared in analogy to example 4A starting from(2R)-5-(Benzyloxy)-2-[(tert-butoxycarbonyl)amino]-5-oxopentanoic acid.

HPLC (Method 7): R_(t)=4.4 min;

LC-MS (Method 11): R_(t)=1.37 min; MS (ESIpos): m/z=390 (M+H)⁺.

Example 11A2-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}sulfanyl)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylglycinate trifluoroacetate

The title compound is prepared in analogy to example 2A starting from2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxyethoxy)phenyl]pyridine-3,5-dicarbonitrileand Boc-glycine.

HPLC (Method 7): R_(t)=5.1 min;

LC-MS (Method 13): R_(t)=1.13 min; MS (ESIpos): m/z=577 (M+H)⁺.

Example 12A2-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}sulfanyl)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylL-phenylalaninate trifluoroacetate

The title compound is prepared in analogy to example 2A starting from2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxyethoxy)phenyl]pyridine-3,5-dicarbonitrileand Boc-L-phenylalanine.

HPLC (Method 7): R_(t)=5.1 min;

LC-MS (Method 13): R_(t)=1.13 min; MS (ESIpos): m/z=577 (M+H)⁺.

Exemplary Embodiments Example 12-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylL-lysyl-L-valinate dihydrochloride

1.5 g (1.77 mmol) of the compound from example 1A, 2.36 g (5.31 mmol) of2,5-dioxopyrrolidin-1-yl-N²,N⁶-bis(tert-butoxycarbonyl)-L-lysinate and1.5 ml of N,N-diisopropylethylamine are mixed in 20 ml of DMF andstirred at room temperature overnight. The solvent is then removed invacuo, and the residue is purified by flash chromatography on silica gelwith dichloromethane/ethyl acetate as eluent (gradient 3:1→2:1). Theappropriate fractions are combined, and the solvent is removed in vacuo.After the residue has been dried under high vacuum, 1.2 g (66% oftheory) of the protected intermediate remain.

HPLC (Method 7): R_(t)=6.7 min.

1.2 g (1.27 mmol) of the resulting intermediate are taken up in 3 ml ofdichloromethane and, while stirring, 50 ml of a saturated solution ofhydrogen chloride in dichloromethane are added. The mixture is stirredat RT for 30 min, during which the target product precipitates. Thesolvent is evaporated off and the remaining residue is stirred with 70ml of diethyl ether. It is filtered off, and the residue on the filteris washed with diethyl ether and then dried under high vacuum. 893 mg(86% of theory) of the title compound result as colorless crystals.

HPLC (Method 7): R_(t)=5.1 min;

LC-MS (Method 12): R_(t)=1.47 min; MS (ESIpos): m/z=747 (M+H)⁺;

¹H-NMR (400 MHz, DMSO-d₆): δ=0.94 and 0.95 (2d, 6H), 1.4 (m, 2H), 1.55(m, 2H), 1.75 (m, 2H), 2.14 (m, 1H), 2.7-2.8 (m, 2H), 3.95 (m, 1H),4.3-4.5 (m, 2H), 4.65 (s, 2H), 7.12 (d, 2H), 7.51 (d, 2H), 7.58 (d, 2H),7.95 (d, 2H), 7.97 (s, 1H), 8.0 (m, 2H), 8.3 (m, 2H), 8.8 (d, 1H).

Example 22-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylβ-alanyl-L-valinate hydrochloride

0.1 g (0.12 mmol) of the compound from example 1A, 0.051 g (0.18 mmol)of 2,5-dioxopyrrolidin-1-yl N-(tert-butoxycarbonyl)-β-alaninate and 82μl of N,N-diisopropylethylamine are mixed in 6 ml of DMF and stirred atroom temperature overnight. The solvent is then removed in vacuo, andthe residue is purified by flash chromatography on silica gel withdichloromethane/ethyl acetate as eluent (gradient 4:1→3:1→2:1). Theappropriate fractions are combined, and the solvent is removed in vacuo.After the residue has been dried under high vacuum, 0.052 g (56% oftheory) of the protected intermediate remains.

HPLC (Method 7): R_(t)=6.3 min.

0.05 g (0.063 mmol) of the resulting intermediate are taken up in 1 mlof dichloromethane and, while stirring, 15 ml of a saturated solution ofhydrogen chloride in dichloromethane are added. The mixture is stirredat RT for 3 h, during which the target compound precipitates. Thesolvent is evaporated off and the remaining residue is stirred with 10ml of diethyl ether. It is filtered off, and the residue on the filteris washed with diethyl ether and then dried under high vacuum. 41 mg(85% of theory) of the title compound result as colorless crystals.

HPLC (Method 7): R_(t)=5.2 min;

LC-MS (Method 12): R_(t)=2.1 min; MS (ESIpos): m/z=690 (M+H)⁺;

¹H-NMR (400 MHz, DMSO-d₆): δ=0.89 and 0.9 (2d, 6H), 2.04 (m, 2H), 2.5(m, 2H), 2.9-3.0 (m, 2H), 4.2 (m, 1H), 4.25 (m, 2H), 4.35-4.5 (m, 2H),4.67 (s, 2H), 7.12 (d, 2H), 7.5 (d, 2H), 7.57 (d, 2H), 7.8 (m, 2H), 7.94(s, 1H), 7.95 (d, 2H), 8.5 (d, 1H).

Example 32-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylL-arginyl-L-valinate dihydrochloride

2.24 g (4.72 mmol) ofN²,N⁵-bis(tert-butoxycarbonyl)-N⁵-{[(tert-butoxycarbonyl)amino](imino)-methyl}-L-ornithine,0.96 g (7.08 mmol) of 1-hydroxy-1H-benzotriazol hydrate and 1.09 g (5.66mmol) of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlorideare mixed in 200 ml of DMF. After stirring for 30 min, 2 g (2.36 mmol)of the compound from example 1A and 1.65 ml of N,N-diisopropylethylamineare added, and the mixture is stirred at room temperature overnight. Itis then concentrated and the remaining residue is stirred with water. Itis filtered off with suction, and the residue is purified by flashchromatography on silica gel with dichloromethane/ethyl acetate aseluent (gradient 4:1→3:1). The appropriate fractions are combined andthe solvent is removed in vacuo. After the residue has been dried underhigh vacuum, 1.12 g (44% of theory) of the protected intermediateremain.

HPLC (Method 7): R_(t)=6.1 min.

1.12 g (1.04 mmol) of the intermediate are taken up in 10 ml ofdichloromethane, mixed with 10 ml of anhydrous trifluoroacetic acid andstirred at RT overnight. The mixture is then concentrated and theresidue is stripped off twice with THF. It is filtered off, and theresidue on the filter is taken up in a mixture of 25 ml of THF and 5 mlof methanol. While stirring, 20 ml of a 2 M solution of hydrogenchloride in diethyl ether are added. After brief further stirring, theprecipitate which has separated out is filtered off with suction, andthe residue on the filter is washed with diethyl ether. After dryingunder high vacuum, 920 mg (99% of theory) of the title compound remainas colorless crystals.

HPLC (Method 7): R_(t)=5.1 min;

LC-MS (Method 10): R_(t)=1.7 min; MS (ESIpos): m/z=775 (M+H)⁺;

¹H-NMR (400 MHz, DMSO-d₆): δ=0.95 and 0.97 (2d, 6H), 1.6 (m, 2H), 1.75(m, 2H), 2.14 (m, 1H), 3.25 (m, 2H), 4.05 (m, 1H), 4.25 (t, 1H), 4.3 (m,2H), 4.4-4.5 (2m, 2H), 4.65 (s, 2H), 7.12 (d, 2H), 7.48 (d, 2H), 7.58(d, 2H), 7.95 (m, 3H), 8.4 (m, 2H), 8.9 (d, 1H).

Example 42-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylL-lysyl-L-alaninate dihydrochloride

1.2 g (1.7 mmol) of the compound from example 2A, 1.13 g (2.55 mmol) of2,5-dioxopyrrolidin-1-yl-N²,N⁶-bis(tert-butoxycarbonyl)-L-lysinate and1.5 ml of N,N-diisopropylethylamine are mixed in 40 ml of DMF andstirred at room temperature overnight. The mixture is then concentratedand the residue is partitioned between ethyl acetate and water. Theorganic phase is separated off and successively extracted twice with 5%strength citric acid and twice with 5% strength sodium bicarbonatesolution. The organic phase is then concentrated and the residue ispurified by flash chromatography on silica gel withdichloromethane/ethyl acetate as eluent (gradient 3:1→2:1).

The appropriate fractions are combined and the solvent is removed invacuo. The residue is stirred with 50 ml of diethyl ether and 50 ml ofpentane and filtered off with suction. After the residue has been driedunder high vacuum, 1.14 g (73% of theory) of the protected intermediateremain.

HPLC (Method 7): R_(t)=6.4 min;

LC-MS (Method 11): R_(t)=2.64 min; MS (ESIpos): m/z=919 (M+H)⁺.

1.14 g (1.24 mmol) of the intermediate are taken up in 10 ml ofdichloromethane and, while stirring, 60 ml of a saturated solution ofhydrogen chloride in dichloromethane are added. The mixture is stirredat RT overnight, during which the target compound precipitates. Thesolvent is concentrated to about one third of the volume, and theresulting suspension is mixed with 200 ml of diethyl ether. The solid isfiltered off, and the residue on the filter is washed with diethyl etherand then dried under high vacuum. 1.0 g (quant.) of the title compoundresults as colorless crystals.

HPLC (Method 7): R_(t)=5.0 min;

LC-MS (Method 10): R_(t)=1.67 min; MS (ESIpos): m/z=719 (M+H)⁺;

¹H-NMR (400 MHz, DMSO-d₆): δ=1.35 (d, 3H), 1.4 (m, 2H), 1.6 (m, 2H),1.75 (m, 2H), 2.75 (m, 2H), 3.8 (m, 1H), 4.25 (m, 2H), 4.3-4.5 (m, 3H),4.63 (s, 2H), 7.12 (d, 2H), 7.48 (d, 2H), 7.58 (d, 2H), 7.9-8.0 (m, 5H),8.3 (m, 2H), 9.05 (d, 1H).

Example 52-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylL-lysyl-L-serinate dihydrochloride

0.58 g (1.675 mmol) of N²,N⁶-bis(tert-butoxycarbonyl)-L-lysine, 0.28 g(1.83 mmol) of 1-hydroxy-1H-benzotriazole hydrate and 0.35 g (1.83 mmol)of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride aremixed in 40 ml of DMF and then 1.01 g (1.52 mmol) of the compound fromexample 3A are added. The mixture is stirred at room temperatureovernight and then poured into a mixture of half-saturated ammoniumchloride solution and dichloromethane. The organic phase is separatedoff, washed successively with water, saturated sodium bicarbonatesolution and saturated sodium chloride solution, dried over magnesiumsulfate, filtered and concentrated. The residue is purified by flashchromatography on silica gel initially with dichloromethane/ethylacetate as eluent (gradient 3:1→2:1); subsequent elution is withdichloromethane/ethyl acetate/methanol (150:50:5). The appropriatefractions are combined and concentrated. After the residue has beendried under high vacuum, 1.23 g (81% of theory) of the protectedintermediate remain.

HPLC (Method 7): R_(t)=6.5 min;

LC-MS (Method 12): R_(t)=2.99 min; MS (ESIpos): m/z=991 (M+H)⁺.

1.216 g (1.48 mmol) of the intermediate are taken up in 6 ml ofdichloromethane, mixed with 6 ml of anhydrous trifluoroacetic acid andstirred at RT overnight. The mixture is then concentrated, and theresidue is stripped off again with dichloromethane. It is filtered off,and the residue on the filter is taken up in a mixture of 25 ml ofdichloromethane and 25 ml of ethyl acetate. While stirring, 20 ml of a 2M solution of hydrogen chloride in diethyl ether are added. After brieffurther stirring, the precipitate which separates out is filtered offwith suction, washed with diethyl ether and dried under high vacuum. Theresidue is then recrystallized from 20 ml of methanol and 20 ml of ethylacetate. The precipitate is again filtered off with suction, washed withethyl acetate and dried under high vacuum. 845 mg (70% of theory) of thetitle compound are obtained as colorless crystals.

HPLC (Method 7): R_(t)=4.9 min;

LC-MS (Method 10): R_(t)=1.62 min; MS (ESIpos): m/z=735 (M+H)⁺.

Example 62-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylN-(2-aminoethyl)-L-α-glutaminate dihydrochloride

1 g (1.92 mmol) of2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxy-ethoxy)phenyl]pyridine-3,5-dicarbonitrile,0.824 g (2.11 mmol) of the compound from example 4A, 0.442 g (2.31 mmol)of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.024g (0.19 mmol) of 4-dimethylaminopyridine are mixed in 40 ml ofdichloromethane and 10 ml of DMF and stirred at room temperatureovernight. The mixture is then poured into a mixture of half-saturatedammonium chloride solution and dichloromethane. The organic phase isseparated off, washed successively with water, saturated sodiumbicarbonate solution and saturated sodium chloride solution, dried overmagnesium sulfate, filtered and concentrated. The residue is purified byflash chromatography on silica gel initially with dichloromethane/ethylacetate (3:1) as eluent; subsequent elution is withdichloromethane/ethyl acetate/methanol (gradient 300:100:5→300:100:10).The appropriate fractions are combined and the solvent is removed invacuo. After the residue has been dried under high vacuum, 1.52 g (89%of theory) of the protected intermediate remain.

HPLC (Method 7): R_(t)=6.0 min;

LC-MS (Method 11): R_(t)=2.54 min; MS (ESIpos): m/z=891 (M+H)⁺.

1.518 g (1.7 mmol) of the intermediate are taken up in 5 ml ofdichloromethane, mixed with 5 ml of anhydrous trifluoroacetic acid, andstirred at RT for 1 h. The mixture is then concentrated, and the residueis again stripped off with dichloromethane. The residue is thendissolved in 20 ml of ethyl acetate. While stirring, 20 ml of a 2 Msolution of hydrogen chloride in diethyl ether are added. Briefsubsequent stirring is followed by filtration with suction, and theresidue on the filter is washed with diethyl ether and dried. 1300 mg(99% of theory) of the title compound are obtained. It is thenrecrystallized from 25 ml of methanol and 25 ml of ethyl acetate. Theprecipitate is again filtered off with suction, washed with ethylacetate and then dried under high vacuum. 1080 mg (83% of theory) of thetitle compound remain.

HPLC (Method 7): R_(t)=4.9 min;

LC-MS (Method 10): R_(t)=1.59 min; MS (ESIpos): m/z=691 (M+H)⁺;

¹H-NMR (400 MHz, DMSO-d₆): δ=2.05 (m, 2H), 2.45 (m, 2H), 2.85 and 2.95(2m, 2H), 3.25 and 3.5 (2m, 2H), 3.7 (m, 1H), 4.3 (m, 2H), 4.4 (m, 2H),4.65 (s, 2H), 7.12 (d, 2H), 7.48 (d, 2H), 7.58 (d, 2H), 7.93 (s, 1H),7.94 (d, 2H), 8.1 (m, 3H), 8.45 (m, 3H), 8.95 (t, 1H).

Example 72-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylN-(2-aminoethyl)-L-glutaminate dihydrochloride

1 g (1.92 mmol) of2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxy-ethoxy)phenyl]pyridine-3,5-dicarbonitrile,0.824 g (2.11 mmol) of the compound from example 5A, 0.442 g (2.31 mmol)of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.024g (0.192 mmol) of 4-dimethylaminopyridine are mixed in 40 ml ofdichloromethane and 10 ml of DMF and stirred at room temperatureovernight. The mixture is then poured into a mixture of half-saturatedammonium chloride solution and dichloromethane. The organic phase isseparated off, washed successively with water, saturated sodiumbicarbonate solution and saturated sodium chloride solution, dried overmagnesium sulfate, filtered and concentrated. The residue isprecipitated from dichloromethane with diethyl ether. The precipitate isfiltered off, washed with diethyl ether and dried under high vacuum. 1.5g (87% of theory) of the protected intermediate remain.

HPLC (Method 7): R_(t)=6.0 min;

LC-MS (Method 10): R_(t)=3.12 min; MS (ESIpos): m/z=891 (M+H)⁺.

1.5 g (1.7 mmol) of the intermediate are taken up in 20 ml ofdichloromethane, mixed with 5 ml of anhydrous trifluoroacetic acid, andstirred at RT for 1 h. The mixture is then concentrated, and the residueis stripped off with toluene several times. The residue is then taken upin 15 ml of dichloromethane, 5 ml of ethyl acetate and 1 ml of methanol.While stirring, 20 ml of a 2 M solution of hydrogen chloride in diethylether are added. Brief subsequent stirring is followed by filtrationwith suction, and the residue on the filter is washed twice with diethylether and dried. The residue is then dissolved in 50 ml of dilutehydrochloric acid (pH 3) and lyophilized. 1265 mg (98% of theory) of thetitle compound remain.

HPLC (Method 7): R_(t)=4.9 min;

LC-MS (Method 11): R_(t)=1.19 min; MS (ESIpos): m/z=691 (M+H)⁺.

Example 82-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylN-(2-aminoethyl)-L-asparaginate dihydrochloride

1 g (1.92 mmol) of2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxy-ethoxy)phenyl]pyridine-3,5-dicarbonitrile,0.794 g (2.12 mmol) of the compound from example 6A, 0.442 g (2.31 mmol)of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.024g (0.192 mmol) of 4-dimethylaminopyridine are mixed in 40 ml ofdichloromethane and 10 ml of DMF and stirred at room temperatureovernight. The mixture is then poured into a mixture of half-saturatedammonium chloride solution and dichloromethane. The organic phase isseparated off, washed successively with water, saturated sodiumbicarbonate solution and saturated sodium chloride solution, dried overmagnesium sulfate, filtered and concentrated. The residue isprecipitated from dichloromethane with diethyl ether. The precipitate isfiltered off, washed with diethyl ether and dried under high vacuum.1.28 g (74% of theory) of the protected intermediate remain.

HPLC (Method 7): R_(t)=6.0 min;

LC-MS (Method 10): R_(t)=3.13 min; MS (ESIpos): m/z=877 (M+H)⁺.

1.28 g (1.46 mmol) of the intermediate are taken up in 20 ml ofdichloromethane, mixed with 5 ml of anhydrous trifluoroacetic acid, andstirred at RT for 1 h. The mixture is then concentrated, and the residueis stripped off several times with toluene. The residue is then taken upin 15 ml of dichloromethane, 5 ml of ethyl acetate and 1 ml of methanol.While stirring, 5 ml of a 2 M solution of hydrogen chloride in diethylether are added. Brief subsequent stirring is followed by filtrationwith suction, and the residue on the filter is washed twice with diethylether and dried. 1096 mg (quant.) of the title compound remain.

HPLC (Method 7): R_(t)=4.9 min;

LC-MS (Method 10): R_(t)=1.58 min; MS (ESIpos): m/z=677 (M+H)⁺.

Example 9(2S)-5-(2-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-3,5-dicyanopyridin-4-yl]phenoxy}ethoxy)-2-[(3-carboxypropanoyl)amino]-5-oxopentanoicacid

0.432 g (2.48 mmol) of 4-tert-butoxy-4-oxobutyric acid, 0.475 g (2.48mmol) of 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochloride,0.380 g (2.48 mmol) of 1-hydroxy-1H-benzotriazole hydrate and 1.59 g(2.254 mmol) of the compound from example 7A are mixed in 70 ml of DMFand stirred at room temperature overnight. The mixture is then pouredinto a mixture of half-saturated ammonium chloride solution and ethylacetate. The organic phase is separated off, washed successively withwater, saturated sodium bicarbonate solution and saturated sodiumchloride solution, dried over magnesium sulfate, filtered andconcentrated. The residue is purified by flash chromatography on silicagel with dichloromethane/ethyl acetate as eluent (gradient10:1→5:1→3:1). The appropriate fractions are combined, and the solventis removed in vacuo. The residue is then purified again in portions bypreparative HPLC (method 6b). The appropriate fractions are combined,and the solvent is removed in vacuo. After the residue has been driedunder high vacuum, 1.63 g (84% of theory) of the protected intermediateremain.

HPLC (Method 7): R_(t)=6.3 min;

LC-MS (Method 11): R_(t)=2.71 min; MS (ESIpos): m/z=861 (M+H)⁺.

1.285 g (1.49 mmol) of the intermediate are taken up in 10 ml ofdichloromethane, mixed with 10 ml of anhydrous trifluoroacetic acid, andstirred at RT for 1 h. The mixture is then concentrated, and the residueis stripped off several times with toluene. The remaining residue isthen stirred with diethyl ether, and the precipitated solid is filteredoff with suction and washed with diethyl ether. After drying under highvacuum, 1.06 g (95% of theory) of the title compound remain.

HPLC (Method 7): R_(t)=5.4 min;

LC-MS (Method 10): R_(t)=2.23 min; MS (ESIpos): m/z=749 (M+H)⁺.

Example 10(2S)-5-(2-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-3,5-dicyanopyridin-4-yl]phenoxy}ethoxy)-2-[(3-carboxypropanoyl)amino]-5-oxopentanoicacid disodium salt

0.5 g (0.667 mmol) of the compound from example 9 is dissolved in 12.5ml of acetonitrile and 62.5 ml of water, and 13 ml of 0.1 N sodiumhydroxide solution are added. After brief stirring, the mixture islyophilized. After drying under high vacuum, 0.53 g (quant.) of thetitle compound remain.

HPLC (Method 7): R_(t)=5.4 min;

LC-MS (Method 11): R_(t)=2.06 min; MS (ESIpos): m/z=749 (M+H)⁺.

Example 112-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}sulfanyl)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylL-lysyl-L-leucinate dihydrochloride

0.928 g (2.68 mmol) of N²,N⁶-bis(tert-butoxycarbonyl)-L-lysine, 0.362 g(2.68 mmol) of 1-hydroxy-1H-benzotriazole hydrate and 0.411 g (2.144mmol) of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlorideare mixed in 200 ml of DMF. Then 1.335 g (1.787 mmol) of the compoundfrom example 8A and 935 μl of N,N-diisopropylethylamine are added, andthe mixture is stirred at room temperature overnight. It is subsequentlyconcentrated in vacuo, and the residue is taken up in ethyl acetate andextracted successively with water, with 5% strength citric acid andtwice with 5% strength sodium bicarbonate solution. The organic phase isconcentrated and the residue is purified by flash chromatography onsilica gel with dichloromethane/ethyl acetate as eluent (gradient3:1→2:1). The appropriate fractions are combined, and the solvent isremoved in vacuo. After the residue has been dried under high vacuum,1.39 g (81% of theory) of the protected intermediate remain.

HPLC (Method 7): R_(t)=6.8 min.

1.385 g (1.44 mmol) of the resulting intermediate are taken up in 20 mlof dichloromethane and, while stirring, 50 ml of a saturated solution ofhydrogen chloride in dichloromethane are added. The mixture is stirredat RT for 1 h, during which the target product precipitates. The solventis evaporated off and the remaining residue is mixed with 70 ml ofpentane, briefly stirred and then filtered off with suction. Dryingunder high vacuum results in 1.17 g (97% of theory) of the titlecompound.

HPLC (Method 7): R_(t)=5.17 min;

LC-MS (Method 10): R_(t)=1.76 min; MS (ESIpos): m/z=761 (M+H)⁺.

Example 122-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}sulfanyl)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylL-lysyl-D-alaninate dihydrochloride

0.59 g (1.702 mmol) of N²,N⁶-bis(tert-butoxycarbonyl)-L-lysine, 0.345 g(2.553 mmol) of 1-hydroxy-1H-benzotriazole hydrate and 0.391 g (2.042mmol) of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlorideare mixed in 24 ml of DMF. After stirring at RT for 5 min, 1.2 g (1.702mmol) of the compound from example 9A and 1.5 ml ofN,N-diisopropylethylamine are added, and the mixture is stirred at roomtemperature overnight. It is then concentrated, and the residue ispartitioned between 500 ml of ethyl acetate and 500 ml of water. Theorganic phase is separated off and extracted successively three timeswith 5% strength citric acid and three times with 10% strength sodiumbicarbonate solution. The organic phase is then concentrated and theresidue is purified by flash chromatography on silica gel withdichloromethane/ethyl acetate as eluent (gradient 2:1→1:1). Theappropriate fractions are combined and the solvent is removed in vacuo.The residue is stirred with 30 ml of ethyl acetate and then mixed with100 ml of diethyl ether. The product is filtered off with suction, andthe remaining residue is washed with diethyl ether. After drying underhigh vacuum, 0.773 g (49% of theory) of the protected intermediateremain.

HPLC (Method 7): R_(t)=6.1 min.

0.74 g (0.805 mmol) of the resulting intermediate are taken up in 200 mlof dichloromethane. Hydrogen chloride gas is passed into this solutionwhile stirring. The deprotected title compound precipitates during this.Stirring is continued at RT, and the reaction is complete after 1 h. Themixture is concentrated to half the volume in vacuo, and the precipitateis filtered off. The residue on the filter is washed with diethyl etherand then dried under high vacuum at 100° C. 539 mg (85% of theory) ofthe title compound are obtained in this way as colorless crystals.

HPLC (Method 7): R_(t)=5.0 min;

LC-MS (Method 13): R_(t)=1.04 min; MS (ESIpos): m/z=719 (M+H)⁺;

¹H-NMR (400 MHz, DMSO-d₆): δ=1.3 (d, 3H), 1.35 (m, 2H), 1.52 (m, 2H),1.75 (m, 2H), 2.75 (m, 2H), 3.8 (m, 1H), 4.3 (m, 2H), 4.3-4.5 (m, 3H),4.63 (s, 2H), 7.12 (d, 2H), 7.48 (d, 2H), 7.58 (d, 2H), 7.8-8.0 (m, 5H),8.25 (m, 2H), 9.0 (d, 1H).

Example 132-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}sulfanyl)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylN-(2-aminoethyl)-D-α-glutaminate dihydrochloride

0.522 g (1.0 mmol) of2-amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}thio)-4-[4-(2-hydroxyethoxy)phenyl]pyridine-3,5-dicarbonitrile,0.430 g (1.104 mmol) of the compound from example 10A, 0.231 g (1.204mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and0.012 g (0.1 mmol) of 4-dimethylaminopyridine are mixed in 20 ml ofdichloromethane and 5 ml of DMF and stirred at room temperatureovernight. The mixture is then poured into a mixture of half-saturatedammonium chloride solution and dichloromethane. The organic phase isseparated off, washed successively with water, saturated sodiumbicarbonate solution and saturated sodium chloride solution, dried overmagnesium sulfate, filtered and concentrated. The residue is purified byflash chromatography on silica gel firstly with dichloromethane/ethylacetate (3:1) as eluent; subsequent elution is withdichloromethane/ethyl acetate/methanol (gradient 300:100:5→300:100:10).The appropriate fractions are combined and the solvent is removed invacuo. After the residue has been dried under high vacuum, 0.711 g (79%of theory) of the protected intermediate remains.

HPLC (Method 7): R_(t)=6.0 min;

LC-MS (Method 13): R_(t)=1.54 min; MS (ESIpos): m/z=891 (M+H)⁺.

0.711 g (0.798 mmol) of the resulting intermediate are taken up in 4 mlof dichloromethane, mixed with 4 ml of anhydrous trifluoroacetic acid,and stirred at RT for 1 h. The mixture is then concentrated, and theresidue is again stripped off with dichloromethane. The residue is thendissolved in 50 ml of ethyl acetate. While stirring, 20 ml of a 2 Msolution of hydrogen chloride in diethyl ether are added. Briefsubsequent stirring is followed by filtration with suction, and theresidue on the filter is washed with diethyl ether and dried. 590 mg(97% of theory) of the title compound are obtained.

HPLC (Method 7): R_(t)=4.9 min;

LC-MS (Method 11): R_(t)=1.22 min; MS (ESIpos): m/z=691 (M+H)⁺;

¹H-NMR (400 MHz, DMSO-d₆): δ=2.05 (m, 2H), 2.45 (m, 2H), 2.85 and 2.95(2m, 2H), 3.25 and 3.5 (2m, 2H), 3.7 (m, 1H), 4.3 (m, 2H), 4.4 (m, 2H),4.65 (s, 2H), 7.12 (d, 2H), 7.48 (d, 2H), 7.58 (d, 2H), 7.93 (s, 1H),7.94 (d, 2H), 8.1 (m, 3H), 8.45 (m, 3H), 8.93 (t, 1H).

Example 142-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}sulfanyl)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylL-arginyl-L-alaninate dihydrochloride

0.269 g (0.567 mmol) ofN⁵-[N,N′-bis(tert-butoxycarbonyl)carbamidoyl]-N²-(tert-butoxycarbonyl)-L-ornithine,0.115 g (0.851 mmol) of 1-hydroxy-1H-benzotriazole hydrate and 0.131 g(0.681 mmol) of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride are mixed in 20 ml of DMF. After stirring for 30 min, 0.2g (0.284 mmol) of the compound from example 2A and 200 μl ofN,N-diisopropylethylamine are added, and the mixture is stirred at roomtemperature overnight. It is then concentrated in vacuo, and the residueis taken up in dichloromethane and extracted successively with 5%strength citric acid, 5% strength sodium bicarbonate solution and water.The organic phase is concentrated and the residue is purified by flashchromatography on silica gel with dichloromethane/ethyl acetate (3:1) aseluent. The appropriate fractions are combined, and the solvent isremoved in vacuo. After the residue has been dried under high vacuum,0.179 g (60% of theory) of the protected intermediate remains.

HPLC (Method 7): R_(t)=5.8 min.

0.178 g (0.17 mmol) of the resulting intermediate are taken up in 30 mlof a saturated solution of hydrogen chloride in dichloromethane andstirred at RT overnight. The mixture is concentrated to half the volumein vacuo, and the resulting precipitate is filtered off. The residue onthe filter is washed with diethyl ether and then dried under highvacuum. 119 mg (82% of theory) of the title compound are obtained ascolorless crystals in this way.

HPLC (Method 7): R_(t)=4.9 min;

LC-MS (Method 10): R_(t)=1.58 min; MS (ESIpos): m/z=747 (M+H)⁺;

¹H-NMR (400 MHz, DMSO-d₆): δ=1.35 (d, 3H), 1.55 (m, 2H), 1.75 (m, 2H),3.25 (m, 2H), 3.85 (m, 1H), 4.25 (m, 2H), 4.3-4.5 (m, 3H), 4.65 (s, 2H),7.12 (d, 2H), 7.48 (d, 2H), 7.58 (d, 2H), 7.75 (t, 1H), 7.93 (s, 1H),7.94 (d, 2H), 8.3 (m, 3H), 9.1 (d, 1H).

Example 152-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}sulfanyl)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylL-histidyl-L-alaninate dihydrochloride

0.145 g (0.567 mmol) of N-(tert-butoxycarbonyl)-L-histidine, 0.115 g(0.851 mmol) of 1-hydroxy-1H-benzotriazol hydrate and 0.131 g (0.681mmol) of N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochlorideare mixed in 20 ml of DMF. After stirring for 30 min, 0.2 g (0.284 mmol)of the compound from example 2A and 200 μl of N,N-diisopropylethylamineare added, and the mixture is stirred at room temperature overnight. Itis then concentrated in vacuo, and the residue is taken up indichloromethane and extracted successively with 5% strength citric acid,5% strength sodium bicarbonate solution and water. The organic phase isconcentrated, and the residue is purified by flash chromatography onsilica gel with toluene/ethanol (3:1) as eluent. The appropriatefractions are combined, and the solvent is removed in vacuo. After theresidue has been dried under high vacuum, 0.206 g (88% of theory) of theprotected intermediate remains.

HPLC (Method 7): R_(t)=5.3 min.

0.206 g (0.249 mmol) of the resulting intermediate are taken up in 30 mlof a saturated solution of hydrogen chloride in dichloromethane andstirred at RT for 2 h. The mixture is concentrated to half the volume invacuo, and the resulting precipitate is filtered off. The residue on thefilter is washed with diethyl ether and then dried under high vacuum.171 mg (90% of theory) of the title compound are obtained as colorlesscrystals in this way.

HPLC (Method 7): R_(t)=4.8 min;

LC-MS (Method 10): R_(t)=1.59 min; MS (ESIpos): m/z=728 (M+H)⁺;

¹H-NMR (400 MHz, DMSO-d₆): δ=1.35 (d, 3H), 3.15 and 3.25 (2dd, 2H), 4.25(m, 3H), 4.3-4.5 (m, 3H), 4.65 (s, 2H), 7.12 (d, 2H), 7.4 (s, 1H), 7.48(d, 2H), 7.58 (d, 2H), 7.93 (s, 1H), 7.94 (d, 2H), 8.5 (m, 3H), 9.05 (s,1H), 9.2 (d, 1H).

Example 162-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}sulfanyl)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylN-[(2S)-2,4-diaminobutanoyl]-L-alaninate dihydrochloride

0.181 g (0.567 mmol) of (2S)-2,4-bis[(tert-butoxycarbonyl)amino]butyricacid, 0.115 g (0.851 mmol) of 1-hydroxy-1H-benzotriazol hydrate and0.131 g (0.681 mmol) of N-(3-dimethyl-aminopropyl)-N′-ethylcarbodiimidehydrochloride are mixed in 20 ml of DMF. After stirring at RT for 30min, 0.2 g (0.284 mmol) of the compound from example 2A and 200 μl ofN,N-diisopropyl-ethylamine are added, and the mixture is stirred at roomtemperature overnight. It is then concentrated in vacuo, and the residueis taken up in dichloromethane and extracted successively with 5%strength citric acid, 5% strength sodium bicarbonate solution and water.The organic phase is concentrated and the residue is purified by flashchromatography on silica gel with toluene/ethanol (10:1) as eluent. Theappropriate fractions are combined, and the solvent is removed in vacuo.After the residue has been dried under high vacuum, 0.217 g (86% oftheory) of the protected intermediate remains.

HPLC (Method 7): R_(t)=6.3 min.

0.212 g (0.238 mmol) of the resulting intermediate is taken up in 25 mlof a saturated solution of hydrogen chloride in dichloromethane andstirred at RT for 1.5 h. The mixture is concentrated to half the volumein vacuo, and the resulting precipitate is filtered off. The residue onthe filter is washed with diethyl ether and then dried under highvacuum. 171 mg (94% of theory) of the title compound are obtained ascolorless crystals in this way.

HPLC (Method 7): R_(t)=4.8 min;

LC-MS (Method 10): R_(t)=1.56 min; MS (ESIpos): m/z=691 (M+H)⁺.

Example 172-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}sulfanyl)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylL-lysylglycinate dihydrochloride

0.185 g (0.535 mmol) of N²,N⁶-bis(tert-butoxycarbonyl)-L-lysine, 0.108 g(0.803 mmol) of 1-hydroxy-1H-benzotriazole hydrate and 0.123 g (0.642mmol) of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlorideare mixed in 15 ml of DMF and stirred at RT for 5 min. Then 0.37 g(0.535 mmol) of the compound from example 11A and 466 μl ofN,N-diisopropylethylamine are added, and the mixture is stirred at roomtemperature overnight. It is then concentrated in vacuo, and the residueis partitioned between 500 ml of ethyl acetate and 200 ml of water. Theorganic phase is separated off, extracted successively three times with5% strength citric acid and three times with 5% strength sodiumbicarbonate solution and dried over magnesium sulfate. The organic phaseis concentrated, and the residue is stirred with 50 ml of ethyl acetatefor 10 min. Then 100 ml of diethyl ether are slowly added, and theprecipitate is filtered off with suction. After drying under highvacuum, 0.303 g (63% of theory) of the protected intermediate remains.

HPLC (Method 7): R_(t)=6.1 min.

0.279 g (0.308 mmol) of the resulting intermediate is taken up in 120 mlof dichloromethane. Hydrogen chloride gas is passed into this solutionwhile stirring, during which the deprotected title compoundprecipitates. The mixture is then stirred at RT for 1 h. It is thenconcentrated to half the volume in vacuo, and 30 ml of absolute THF areslowly added. After stirring for a further 15 min, the precipitate isfiltered off. The residue on the filter is washed with diethyl ether andthen dried under high vacuum. 212 mg (88% of theory) of the titlecompound are obtained in this way as colorless crystals.

HPLC (Method 7): R_(t)=4.8 min;

LC-MS (Method 10): R_(t)=1.52 min; MS (ESIpos): m/z=705 (M+H)⁺.

Example 182-{4-[2-Amino-6-({[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methyl}sulfanyl)-3,5-dicyanopyridin-4-yl]-phenoxy}ethylL-lysyl-L-phenylalaninate dihydrochloride

0.166 g (0.48 mmol) of N²,N⁶-bis(tert-butoxycarbonyl)-L-lysine, 0.097 g(0.72 mmol) of 1-hydroxy-1H-benzotriazole hydrate and 0.110 g (0.576mmol) of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlorideare mixed in 7 ml of DMF and stirred at RT for 5 min. Then 0.375 g (0.48mmol) of the compound from example 12A and 418 μl ofN,N-diisopropylethylamine are added, and the mixture is stirred at roomtemperature overnight. It is then concentrated in vacuo, and the residueis taken up in 200 ml of ethyl acetate, extracted successively twicewith 10% strength citric acid and twice with 10% strength sodiumbicarbonate solution and dried over magnesium sulfate. The organic phaseis concentrated, the residue is taken up in 10 ml of ethyl acetate, andthe product is precipitated by adding diethyl ether. The precipitate isfiltered off with suction and dried under high vacuum. 0.338 g (71% oftheory) of the protected intermediate remains.

HPLC (Method 7): R_(t)=6.7 min.

0.32 g (0.321 mmol) of the resulting intermediate is taken up in 100 mlof dichloromethane. Hydrogen chloride gas is passed into this solutionwhile stirring, during which the deprotected title compoundprecipitates. The mixture is stirred at RT for a further 1 h and thenconcentrated to half the volume in vacuo, and 10 ml absolute THF areslowly added. After stirring for a further 15 min, the precipitate isfiltered off. The residue on the filter is washed with diethyl ether andthen dried under high vacuum. 188 mg (67% of theory) of the titlecompound are obtained as colorless crystals in this way.

HPLC (Method 7): R_(t)=5.0 min;

LC-MS (Method 11): R_(t)=1.33 min; MS (ESIpos): m/z=795 (M+H)⁺.

B. DETERMINATION OF SOLUBILITY, STABILITY AND LIBERATION BEHAVIOR

a) Determination of the Solubility:

The test substance is suspended in 5% strength aqueous dextrosesolution. This suspension is shaken at room temperature for 24 h. Afterultracentrifugation at 224 000 g for 30 min, the supernatant is dilutedwith DMSO and analysed by HPLC. A two-point calibration plot of the testcompound in DMSO is used for quantification.

HPLC Method for Acids:

Agilent 1100 with DAD (G1315A), quat. Pump (G1311A), autosampler CTC HTSPAL, degasser (G1322A) and column thermostat (G1316A); column:Phenomenex Gemini C18, 5 μm, 50 mm×2 mm; temperature: 40° C.; eluent A:water/phosphoric acid pH 2, eluent B: acetonitrile; flow rate: 0.7ml/min; gradient: 0-0.5 min 85% A, 15% B; ramp 0.5-3 min 10% A, 90% B;3-3.5 min 10% A, 90% B; ramp 3.5-4 min 85% A, 15% B; 4-5 min 85% A, 15%B.

HPLC Method for Bases:

Agilent 1100 with DAD (G1315A), quat. pump (G1311A), autosampler CTC HTSPAL, degasser (G1322A) and column thermostat (G1316A); column:VDSoptilab Kromasil 100 C18, 3.5 μm, 60 mm×2.1 mm; temperature: 30° C.;eluent A: water+5 ml of perchloric acid/liter, eluent B: acetonitrile;flow rate: 0.75 ml/min; gradient: 0-0.5 min 98% A, 2% B; ramp 0.5-4.5min 10% A, 90% B; 4.5-6 min 10% A, 90% B; ramp 6.5-6.7 min 98% A, 2% B;6.7-7.5 min 98% A, 2% B.

The solubilities of representative exemplary embodiments in 5% strengthaqueous dextrose solution are shown in Table 1:

TABLE 1 Example No. Solubility [mg/Liter] 1 >500 3 450 4 >500 5 >5006 >500 7 100 10 390 11 >500 12 >500 13 >500 14 >500 15 450 17 >500 18450

No decomposition of the exemplary compounds in these solutions isobserved.

The solubility of the underlying active substance [compound (A)] in 5%strength aqueous dextrose solution is determined in this test to be <0.1mg/liter.

b) Stability in Buffer at Various pH Values:

0.3 mg of the test substance is weighed into a 2 ml HPLC vial and 0.5 mlof acetonitrile or acetonitrile/DMSO (9:1) is added. The substance isdissolved by putting the sample vessel in an ultrasonic bath for about10 seconds. Then 0.5 ml of the respective buffer solution is added, andthe sample is again treated in the ultrasonic bath.

(Buffer) Solutions Employed:

pH 2: 0.03 mol of citric acid, 0.061 mol of sodium chloride and 0.0082mol of hydrochoric acid ad 1 liter of water;

pH 4: 1 liter of Millipore water is adjusted to pH 4.0 with 1 Nhydrochloric acid;

pH 5: 0.096 mol of citric acid and 0.2 mol of sodium hydroxide ad 1liter of water;

pH 6: 0.06 mol of citric acid and 0.16 mol of sodium hydroxide ad 1liter of water;

pH 7.4: 90.0 g of sodium chloride, 13.61 g of potassium dihydrogenphosphate and 83.35 g of 1 N sodium hydroxide solution are made up to 1liter with water; this solution is then further diluted 1:10 withMillipore water.

pH 8: 0.013 mol of borax and 0.021 mol of hydrochloric acid ad 1 literof water.

5 μl portions of the test solution are analyzed by HPLC for theircontent of unchanged test substance, and of parent substance (A)produced, every hour over a period of 24 hours at 37° C. The percentageareas of the appropriate peaks are used for quantification.

HPLC Method:

Agilent 1100 with DAD (G1314A), binary pump (G1312A), autosampler(G1329A), column oven (G1316A), thermostat (G1330A); column: Kromasil100 C18, 125 mm×4.6 mm, 5 μm; column temperature: 30° C.; eluent A:water+5 ml of perchloric acid/liter, eluent B: acetonitrile; gradient:0-2.0 min 90% A, 10% B; 2.0-18.0 min 64% A, 36% B; 18.0-20.0 min 64% A,36% B; 20.0-21.0 min 10% A, 90% B; 21.0-23.0 min 90% A, 10% B; 23.0-26.0min 90% A, 10% B; flow rate: 2.0 ml/min; UV detection: 294 nm.

The ratios of the peak areas (F) at the respective time points inrelation to the peak areas at the starting time are shown in Table 2 forrepresentative exemplary embodiments:

TABLE 2 % test substance % test substance after 4 h after 24 h Example[F(t = 4 h) × 100/ [F(t = 24 h) × 100/ No. pH F(t = 0 h)] F(t = 0 h)] 14 100 100 1 6 100 100 1 7.4 99 96 1 8 99 90 2 4 100 100 2 7.4 100 100 34 100 100 3 7.4 99 55 4 4 100 100 4 5 100 98 4 6 97 84 4 7.4 48 2 5 4100 100 5 5 100 98 5 6 95 70 6 2 100 100 6 4 100 100 6 5 96 80 6 6 78 256 7.4 8 0 7 4 100 98 7 7.4 51 2 9 4 100 100 9 7.4 100 100 10 4 100 99 107.4 100 99 11 4 100 100 11 7.4 90 55 12 4 99 98 12 7.4 49 2 13 4 100 9913 7.4 24 0 14 4 100 100 14 7.4 68 8

In this test there is found to be a decrease in the content of testsubstance at the same time as an increase in the active ingredientcompound (A).

c) In Vitro Stability in Rat and Human Plasma:

1 mg of the test substance is weighed into a 2 ml HPLC vial, and 1.5 mlof DMSO and 1 ml of water are added. The substance is dissolved byplacing the sample vessel in an ultrasonic bath for about 10 seconds.0.5 ml of rat or human plasma at 37° C. is added to 0.5 ml of thissolution. The sample is shaken, and about 10 μl are removed for a firstanalysis (time point t₀). 4-6 further aliquots are removed forquantification in the period up to 2 hours after the start ofincubation. The sample is kept at 37° C. during the time of the test.Characterization and quantification take place by HPLC.

HPLC Method:

Agilent 1100 with DAD (G1314A), binary pump (G1312A), autosampler(G1329A), column oven (G1316A), thermostat (G1330A); column: Kromasil100 C18, 250 mm×4 mm, 5 μm; column temperature: 30° C.; eluent A:water+5 ml of perchloric acid/liter, eluent B: acetonitrile; gradient:0-8.0 min 53% A, 47% B; 8.0-18.0 min 53% A, 47% B; 18.0-20.0 min 90% A,10% B; 20.0-21.0 min 90% A, 10% B; 21.0-22.5 min 98% A, 2% B; 22.5-25.0min 98% A, 2% B; flow rate: 2 ml/min; UV detection: 294 nm.

Table 3 indicates the respective times for representative exemplaryembodiments at which 50% of the maximum possible amount of activeingredient compound (A) have been produced (t_(50%A)) after incubationwith rat plasma. For the evaluation, the ratio of the peak areas at theindividual time points compared with the starting time point is used ineach case.

TABLE 3 t_(50%A) [min] Example No. in rat plasma 1 60 2 30 3 55 4 1.7 58.0 6 1.1 7 0.5 9 >120 10 >120 12 2 13 0.5 14 0.5

d) i.v. Pharmacokinetics in Wistar Rats:

On the day before administration of the substance, a catheter forobtaining blood is implanted in the jugular vein of the experimentalanimals (male Wistar rats, body weight 200-250 g) under Isofluran®anesthesia.

On the day of the experiment, a defined dose of the test substance isadministered as solution into the tail vein using a Hamilton® glasssyringe (bolus administration, duration of administration <10 s). Bloodsamples (8-12 time points) are taken through the catheter sequentiallyover the course of 24 h after administration of the substance. Plasma isobtained by centrifuging the samples in heparinized tubes. Acetonitrileis added to a defined plasma volume per time point to precipitateproteins. After centrifugation, test substance and, where appropriate,known cleavage products of the test substance in the supernatant aredetermined quantitatively using a suitable LC/MS-MS method.

The measured plasma concentrations are used to calculate pharmacokineticparameters of the test substance and of the active ingredient compound(A) liberated therefrom, such as AUC, C_(max), T_(1/2) (half-life) andCL (clearance).

After i.v. administration of the compound from example 4, from example5, from example 6 and from example 7, these substances were no longerdetectable in plasma even at the first measurement point. Only theactive ingredient (A) was detectable up to the 24-hour time point too.

e) Oral Pharmacokinetics in Wistar Rats:

On the day before administration of the substance, a catheter forobtaining blood is implanted in the jugular vein of the experimentalanimals (male Wistar rats, body weight 200-250 g) under Isofluran®anesthesia.

On the day of the experiment, a defined dose of the test substance isadministered as solution into the stomach by gavage. Blood samples (8-12time points) are taken through the catheter sequentially over the courseof 24 h after administration of the substance. Plasma is obtained bycentrifuging the samples in heparinized tubes. Acetonitrile is added toa defined plasma volume per time point to precipitate proteins. Aftercentrifugation, test substance and, where appropriate, known cleavageproducts of the test substance in the supernatant are determinedquantitatively using a suitable LC/MS-MS method.

The measured plasma concentrations are used to calculate pharmacokineticparameters of the test substance and of the active ingredient compound(A) liberated therefrom, such as AUC, C_(max), T_(1/2) (half-life).

After oral administration of the compound from example 4, from example 5and from example 6, these substances were no longer detectable in plasmaeven at the first measurement point. Only the active ingredient (A) wasdetectable up to the 24-hour time point too.

f) Determination of the Influence on the Heart Rate of AnesthetizedRats:

Male Wistar rats with a body weight above 250 g are employed. In thenight before the experiment, the animals receive no feed but still havefree access to drinking water. Preparation and investigations arecarried out under Trapanal® anesthesia (100 mg/kg i.p.). Injection andinfusion take place through a catheter in the jugular vein, and theblood pressure is recorded via a catheter in the femoral artery(transducer: Braun, Melsungen). After the preparation, the animals areconnected to a continuous infusion of physiological saline solution tocompensate fluid losses. Test substance or placebo solution areadministered as bolus after an equilibration time of about 1 h. Heartrate and arterial blood pressure are recorded during the equilibrationand over a period of at least 30 min after the bolus injection with theaid of a digital evaluation program.

Table 4 lists the maximum heart rate decrease in the first 30 min afteran i.v. bolus of 100 μg/kg of the active substance (A) or of equivalentdosages of representative exemplary embodiments:

TABLE 4 Heart rate decrease Example No. [%] A 24 1 10 4 19 5 12 6 17 715

C. EXEMPLARY EMBODIMENTS OF PHARMACEUTICAL COMPOSITIONS

The compounds of the invention can be converted into pharmaceuticalpreparations in the following ways:

Tablet:

Composition:

100 mg of the compound of the invention, 50 mg of lactose (monohydrate),50 mg of corn starch (native), 10 mg of polyvinylpyrrolidone (PVP 25)(BASF, Ludwigshafen, Germany) and 2 mg of magnesium stearate.

Tablet weight 212 mg. Diameter 8 mm, radius of curvature 12 mm.

Production:

The mixture of the compound of the invention, lactose and starch isgranulated with a 5% strength solution (m/m) of PVP in water. Thegranules are dried and then mixed with the magnesium stearate for 5 min.This mixture is compressed with a conventional tablet press (see abovefor format of the tablet). As guideline, a compressive force of 15 kN isused for the compression.

Oral Suspension:

Composition:

1000 mg of the compound of the invention, 1000 mg of ethanol (96%), 400mg of Rhodigel® (xanthan gum from FMC, Pennsylvania, USA) and 99 g ofwater.

10 ml of oral suspension are equivalent to a single dose of 100 mg ofthe compound of the invention.

Production:

The Rhodigel is suspended in ethanol, and the compound of the inventionis added to the suspension. The water is added while stirring. Themixture is stirred for about 6 hours until the swelling of the Rhodigelis complete.

Oral Solution:

Composition:

500 mg of the compound of the invention, 2.5 g of polysorbate and 97 gof polyethylene glycol 400. A single dose of 100 mg of the compound ofthe invention corresponds to 20 g oral solution.

Production:

The compound of the invention is suspended in a mixture of polyethyleneglycol and polysorbate with stirring. The stirring is continued untilthe compound of the invention has completely dissolved.

i.v. Solution:

The compound of the invention is dissolved in a concentration below thesaturation solubility in a physiologically tolerated solvent (e.g.isotronic saline solution, 5% glucose solution and/or 30% PEG 400solution, in each case adjusted to a pH of 3-5). The solution isoptionally filtered sterile and/or dispensed into sterile andpyrogen-free injection containers.

1. A method of treatment of a cardiovascular disorder selected from thegroup consisting of: hypertension, peripheral and cardiac vasculardisorders, coronary heart disease, coronary restenosis, myocardialinfarction, acute coronary syndrome, acute coronary syndrome with STelevation, acute coronary syndrome without ST elevation, stable andunstable angina pectoris, myocardial insufficiency, princemetal angina,persistent ischemic dysfunction, temporary postischemic dysfunction,heart failure, tachycardia, atrial tachycardia, arrhythmias, atrial andventricular fibrillation, persistent atrial fibrillation, permanentatrial fibrillation, atrial fibrillation with normal left ventricularfunction, atrial fibrillation with impaired left ventricular function,Wolff-Parkinson-White syndrome, disturbances of peripheral blood flow,elevated levels of fibrinogen and of low density LDL, and elevatedconcentrations of plasminogen activator inhibitor 1 (PAI-1) comprisingadministering an effective amount of a compound of the formula (I)

in which R^(A) is a group of the formula

in which * means the point of linkage to the O atom, L¹ and L² areindependently of one another a bond or —CH₂—, R¹, R² and R³ areindependently of one another hydrogen or methyl, R⁴ and R⁶ are identicalor different and are independently of one another hydrogen or the sidegroup of a natural α-amino acid or its homologs or isomers, R⁵ and R⁷are independently of one another hydrogen or methyl, L³ isstraight-chain or branched (C₂-C₄)-alkanediyl which is additionallysubstituted by amino, R⁸, R⁹ and R¹⁰ are independently of one anotherhydrogen or methyl, m is the number 2, 3, 4, 5 or 6, L⁴ isstraight-chain or branched (C₂-C₄)-alkanediyl, which is additionallysubstituted by carboxyl, R¹¹ is hydrogen or methyl, and n is the number1, 2, 3 or 4, or a salt thereof to a human or animal in need thereof. 2.The method of claim 1, wherein in the compound of formula (I) R^(A) is agroup of the formula

in which * means the point of linkage to the O atom, L¹ is a bond, L² isa bond or —CH₂—, R¹ and R³ are independently of one another hydrogen ormethyl, R⁴ is hydrogen, methyl, propan-2-yl, propan-1-yl,2-methylpropan-1-yl, 1-methyl-propan-1-yl, butan-1-yl, benzyl,p-hydroxybenzyl, imidazol-4-ylmethyl, hydroxymethyl, 1-hydroxyethyl,carbamoylmethyl or 2-carbamoylethyl, R⁶ is hydrogen,imidazol-4-ylmethyl, 4-aminobutan-1-yl, 3-aminopropan-1-yl,2-aminoethyl, aminomethyl or 3-guanidinopropan-1-yl, L³ isstraight-chain (C₂-C₄)-alkanediyl which is additionally substituted byamino, R⁸ and R¹⁰ are independently of one another hydrogen or methyl, mis the number 2, 3 or 4, L⁴ is straight-chain (C₂-C₄)-alkanediyl whichis additionally substituted by carboxyl, R¹¹ is hydrogen or methyl, andn is the number 2, 3 or 4 or a salt thereof.
 3. The method of claim 1wherein in the compound of formula (I): R^(A) is a group of the formula

in which * means the point of linkage to the O atom, L¹ and L² are eacha bond, R⁴ is hydrogen, methyl, propan-2-yl, propan-1-yl,2-methylpropan-1-yl, 1-methyl-propan-1-yl, butan-1-yl, benzyl,p-hydroxybenzyl, imidazol-4-ylmethyl, hydroxymethyl, 1-hydroxyethyl,carbamoylmethyl or 2-carbamoylethyl, R⁶ is imidazol-4-ylmethyl,4-aminobutan-1-yl, 3-aminopropan-1-yl, 2-aminoethyl, aminomethyl or3-guanidinopropan-1-yl, L³ is a group of the formula —CH(NH₂)—CH₂—,—CH₂—CH(NH₂)—, —CH₂—CH(NH₂)—CH₂—, —CH(NH₂)—CH₂—CH₂— or—CH₂—CH₂—CH(NH₂)—, m is the number 2, 3 or 4, L⁴ is a group of theformula **—CH₂—CH(COOH)— or **—CH₂—CH₂—CH(COOH)—, in which ** representsthe point of linkage to the adjoining carbonyl group, and n is thenumber 2 or 3, or a salt thereof.
 4. The method of claim 1, wherein inthe compound of formula (I) R^(A) is a group of the formula

in which * means the point of linkage to the O atom, L¹ and L² are eacha bond, R⁴ is hydrogen, methyl, propan-2-yl, 2-methylpropan-1-yl,benzyl, hydroxymethyl or 1-hydroxyethyl, and R⁶ is imidazol-4-ylmethyl,4-aminobutan-1-yl, 3-aminopropan-1-yl, 2-aminoethyl, aminomethyl or3-guanidinopropan-1-yl, or a salt thereof.
 5. The method of claim 1,wherein in the compound of formula (I) R^(A) is a group of the formula

in which * means the point of linkage to the O atom, L³ is a group ofthe formula —CH(NH₂)—CH₂—, —CH₂—CH(NH₂)—, —CH(NH₂)—CH₂—CH₂— or—CH₂—CH₂—CH(NH₂)—, and m is the number 2 or 3, or a salt thereof. 6-12.(canceled)
 13. The method of claim 1, wherein the compound of formula(I) is a compound having the formula:


14. The method of claim 1, wherein the compound of formula (I) is acompound having the formula:


15. The method of claim 1, wherein the compound of formula (I) is acompound having the formula


16. The method of claim 1, wherein the compound of formula (I) is acompound having the formula


17. The method of claim 1, wherein the compound of formula (I) is acompound having the formula


18. The method of claim 1, wherein the compound of formula (I) is acompound having the formula


19. The method of claim 1, wherein the compound of formula (I) is acompound having the formula


20. The method of claim 1, wherein the cardiovascular disorder isselected from the group consisting of: hypertension, coronary heartdisease, acute coronary syndrome, angina pectoris, heart failure,myocardial infarction and atrial fibrillation.